Chimeric nucleic acid molecules with non-aug translation initiation sequences and uses thereof

ABSTRACT

The present disclosure relates to nucleic acid vaccine compositions and methods for preventing or treating pathological conditions, such as cancer or infectious disease. Further, the disclosure provides methods for more efficient production of antigens via mRNA containing one or more non-conventional start codons to promote multiplex initiation of translation in eukaryotic cells.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 890079_401C4_SEQUENCE_LISTING.txt. The text fileis 210 KB, was created on Jul. 13, 2016, and is being submittedelectronically via EFS-Web.

BACKGROUND

The immune system can be categorized into innate immunity, whichinvolves numerous cellular and soluble factors that respond to allforeign challenges, and adaptive immunity, which responds specificallyto precise epitopes from foreign or abnormal agents. The adaptive immuneresponse includes a humoral arm, which involves the production ofantibodies by B lymphocytes, and a cellular arm, which involves thekiller activity of cytotoxic T lymphocytes (CTLs). A key mechanism fordetecting and eliminating abnormal cells by the adaptive immune responseis surveillance by CTLs. Abnormal cells may be those infected with avirus, parasite or bacteria, or those that have undergone a tumorigenictransformation.

Cells naturally produce a repertoire of peptides from essentially anycellular translation product that has been marked for elimination (e.g.,ubiquitination), which results in presentation of peptide/majorhistocompatibility complex (MHC; in humans known as human leukocyteantigen or HLA) class I complexes on their surface. A ubiquitinatedprotein is targeted to the proteasome for proteolysis, producing smallerpeptides that may be recognized by transporter associated with antigenpresentation (TAP) proteins that are localized in the endoplasmicreticulum. TAP is a heterodimer that moves small peptides from thecytosol into the endoplasmic reticulum where they bind to HLA/MHCmolecules to form a peptide/HLA complex. The peptide/HLA complex is thentrafficked to the cell surface.

T cell receptors (TCRs) on the surface of circulating CTLs probe thepeptide/MHC complexes for the presence of foreign peptides, such asviral proteins or tumor specific proteins, which will trigger a T celldirected immune response. Cells can present tens of thousands ofdistinct peptides in the context of MHC molecules as potential ligandsfor the TCR, although the quantity of each peptide will be very low.Nonetheless, CTLs are very sensitive probes for peptides displayed byMHC class I. By some estimates, only three copies of an antigenicpeptide are sufficient to target cells for lysis (Purbhoo et al., Nat.Immunol. 5:524, 2004).

Vaccines have had a profound and long lasting effect on world health.Smallpox has been eradicated, polio is near elimination, and diseasessuch as diphtheria, measles, mumps, pertussis, and tetanus arecontained. Gene therapy and nucleic acid immunization are promisingapproaches for the treatment and prevention of both acquired andinherited diseases (Li et al., J. Biotechnol. 162:171, 2012). Thesetechniques involve the administration of a desired nucleic acid vaccinedirectly into a subject in vivo, or by transfecting a subject's cells ortissues ex vivo and reintroducing the transformed material into thesubject. Each of these techniques requires efficient expression of anucleic acid molecule in the transfected cell, which may be affected byseveral factors, to provide a sufficient amount of a therapeutic orantigenic gene product. Alternatively, antigenic peptides that aredefined T cell epitopes may be administered directly to form productivepeptide/MHC complexes and stimulate a T cell response.

Current vaccines, however, address only a handful of the infections orcancers suffered by people and domesticated animals. Common infectiousdiseases for which there are no vaccines cost the United States aloneabout $120 billion per year (Robinson et al., American Acad. Microbiol.,1996). In first world countries, emerging infections such asimmunodeficiency viruses, as well as reemerging diseases like drugresistant forms of tuberculosis, pose new threats and challenges forvaccine development. The need for both new and improved vaccines is evenmore pronounced in third world countries where effective vaccines areoften unavailable or cost-prohibitive.

In view of the limitations associated with current vaccines, there is aneed in the art for alternative compositions and methods useful for moreefficient and manageable vaccines and vaccinations. The presentdisclosure meets such needs, and further provides other relatedadvantages.

BRIEF SUMMARY

In some embodiments, the present disclosure provides a chimeric nucleicacid molecule, comprising a multiplex translation initiation (MTI)sequence and a nucleic acid molecule encoding an antigen, an antigenicepitope, or a combination thereof, wherein the MTI comprises at leastone non-AUG translation initiation site that mediates translationinitiation of the antigen, antigenic epitope, or combination thereof.

In certain embodiments, the present disclosure provides a vector,comprising a multiplex translation initiation (MTI) sequence and anucleic acid molecule encoding an antigen, an antigenic epitope, or acombination thereof, wherein the MTI comprises at least one non-AUGtranslation initiation site that mediates translation initiation of theantigen, antigenic epitope, or combination thereof.

In some embodiments, the present disclosure provides a cell, comprisinga chimeric nucleic comprising a multiplex translation initiation (MTI)sequence and a nucleic acid molecule encoding an antigen, an antigenicepitope, or a combination thereof, wherein the MTI comprises at leastone non-AUG translation initiation site that mediates translationinitiation of the antigen, antigenic epitope, or combination thereof.

In further embodiments, the present disclosure provides a cellcomprising a vector, comprising a chimeric nucleic comprising amultiplex translation initiation (MTI) sequence and a nucleic acidmolecule encoding an antigen, an antigenic epitope, or a combinationthereof, wherein the MTI comprises at least one non-AUG translationinitiation site that mediates translation initiation of the antigen,antigenic epitope, or combination thereof.

In some embodiments, the present disclosure provides a method ofeliciting a cellular immune response, comprising administering to asubject an effective amount of an immunization composition comprising anucleic acid molecule according as described in embodiments herein, anantigen encoded by a nucleic acid molecule as described in embodimentsherein, or both, thereby eliciting a cellular immune response.

In some embodiments, the present disclosure provides a method ofeliciting a cellular immune response, comprising administering to asubject an effective amount of a cell comprising a nucleic acid moleculeas described in embodiments herein, or a vector as described inembodiments herein, thereby eliciting a cellular immune response,thereby eliciting a cellular immune response.

In further embodiments, the present disclosure provides a method ofeliciting a cellular immune response, comprising (a) administering to asubject an effective amount of an antigen immunization compositioncomprising one or more antigens encoded by any one of the nucleic acidmolecules as described in embodiments herein, and (b) administering tothe subject an effective amount of a nucleic acid molecule immunizationcomposition comprising a nucleic acid molecule as described inembodiments herein or a vector as described in embodiments herein.

In in yet further embodiments, the present disclosure provides a methodof eliciting an cellular immune response, comprising (a) administeringto a subject an effective amount of a nucleic acid molecule immunizationcomposition comprising a nucleic acid molecule as described inembodiments herein, (b) allowing a time sufficient to generate aninitial immune response, and (c) administering to the subject a secondeffective amount of a nucleic acid molecule immunization compositioncomprising a nucleic acid molecule as described in embodiments herein,or an effective amount of an antigen immunization composition.

In some embodiments, the present disclosure provides a method oftreating breast cancer, comprising administering to a subject aneffective amount of nucleic acid molecule immunization compositioncomprising a comprising a multiplex translation initiation (MTI)sequence and a nucleic acid molecule encoding an antigen, an antigenicepitope, or a combination thereof, wherein the MTI comprises at leastone non-AUG translation initiation site that mediates translationinitiation of the antigen, antigenic epitope, or combination thereof,and the antigenic epitope comprises a peptide from folate receptoralpha, HER2/Neu, or any combination thereof.

In some embodiments, the present disclosure provides a method oftreating cancer, comprising administering to a subject an effectiveamount of a nucleic acid immunization composition comprising a nucleicacid molecule as described in embodiments herein, wherein the one ormore antigens encoded the nucleic acid molecule comprise an antigenhaving an oncogenic mutation identified in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an exemplary DNA vaccine expression vector. A) Thevaccine vector contains full-length, codon-optimized TAP 1 and TAP 2driven by an SV40 promoter and the MTI-peptide antigen array (PAA) underthe control of the CMV promoter. B) Overexpression of the PAA isachieved through the application of three non-classical translationinitiation sites (CUG) that can be found in the MTI. These CUG and AUGsites mediate nuclear and cytosolic expression respectively, and can bealtered as needed. The PAA is flanked by Xba1 sites facilitatinginsertion and excision of various PAAs. C) Peptide sequences in theA*0201 PAA are depicted (SEQ ID NO.: 204). A2 binding peptides arepresented in bold font. Each peptide is flanked by 3-4 amino acidresidues at the NH2 and COOH ends to retain the natural proteasomeprocessing sites for each peptide. Each peptide is also separated by the(G₄S)₂ spacer. This specific PAA also includes an Influenza M1 proteinas a control, and a C-terminal VSVG protein epitope tag (SEQ ID NO.: 14)for evaluating epitope expression.

FIG. 2 depicts immunoprecipitation detection of TAP1 and MTI-PAA. A)Cell extracts from TAP1-transfected COS cells were immunoprecipitatedwith goat anti-V5 antibody (the NH2-terminal tag on recombinant TAP1)and protein G agarose, separated by 12% SDS-PAGE, transferred to PVDF,and immunoblotted with rabbit anti-V5 antibody. B) Cell extracts fromMTI-PAA transfected COS cell were heparin sepharose (HS) purified,separated by 12% PAGE, transferred, and immunoblotted with goatanti-FGF2 antibodies.

FIG. 3 depicts immunofluorescence micrographs of expression of TAP1 inCOS cells using anti-V5 and anti-TAP1 antibodies.

FIG. 4 depicts immunofluorescence micrographs of expression of MTI-PAA.Staining was performed using an anti-VSV antibody directed against theC-terminal portion of the MTI-PAA.

FIG. 5 depicts the immunoprecipitation detection of PAA products in thepresence or absence of proteasome activity. HEK cells were transfectedwith pcDNA3PAA or control. Immunoprecipitation of expression products inthe presence of the proteasome inhibitor MG132 showed the expression ofthe predicted translation products of MTI-PAA.

FIG. 6 depicts T cell recognition of PAA-expressing targets. Splenocytesfrom peptide-vaccinated (#22, #25, #28, #30, HBV core Ag: emulsified inincomplete Freund's adjuvant (IFA)) mice were incubated with THP-1 cells(column 1), THP-1 cells pulsed with 1 ug/ml of each peptide (p22, p25,p28, p30) (column 2), or were transfected with MTI-PAA expression vectorfor 18 hours (column 3).

FIG. 7 depicts T cell reactivity, measured by ELISPOT, against a seriesof stably transfected HEK cell lines. The cell lines weresuper-transfected as described. The first pair of bars correspond to HEKcells stably transfected with a clone expressing Tap1 (white/clear bar).These cells were super-transfected with an expression vector encoding asmall pox PAA (diagonal line bar). Moving left to right, the next fourpairs of bars represent four independent stably transfected HEK celllines expressing Tap1. Each Tap1 transfected cell line wassuper-transfected with a PolyStart™-PAA encoding small pox antigenicpeptides. The diagonal lined bars show elevated T-cell reactivity,indicating proteasome processing of a small pox PAA. The two right mostbars are negative controls (clear/white is non-transfected normal HEKcells and HEK cells pulsed with small pox peptides only).

FIG. 8 depicts results from IFNγ ELISPOT assays measuring the immuneresponse in HLA-A2 mice after a two-dose vaccination regimen. Peptideresponses are reported as the fold increase (in IFNγ spot forming units)in response to target cells expressing peptide immunogen over responseto target cells expressing an unrelated peptide. All responses shown aresignificant (p<0.05) except p19 and p25.

FIG. 9 depicts an immune response after peptide vaccination. HLA-A2 micewere vaccinated twice with peptides #22, #25, #28 emulsified in IFA.Immune responses were measured against syngeneic splenocytes pulsed withpooled peptide cocktail (peptides #22, #25, #28: 1 uM each)(cross-hatched bars) or unpulsed (white bars).

FIG. 10 depicts the effect of peptide-vaccine on survival after lethalintranasal challenge with VACV-WR. Kaplan-Meier survival curve forunvaccinated mice (broken line) and peptide-vaccinated (#22, #25, #28,#30) mice (solid line) following lethal intranasal challenge withVACV-WR (1×10⁶ pfu). Significance was assessed using the log-rank test.

DETAILED DESCRIPTION

In some aspects, the present disclosure provides a chimeric nucleic acidmolecule comprising a multiplex translation initiation (MTI) sequenceand a nucleic acid molecule encoding one or more antigens, antigenicepitopes, or a combination thereof (e.g., polyantigen array or PAA),wherein the multiplex translation sequence comprises at least onenon-AUG translation initiation site that mediates translation initiationof the one or more antigens, antigenic epitopes, or a combinationthereof.

In certain embodiments, the present disclosure provides a method forprime and boost or multiple antigenic challenges to elicit a robustimmune response that results in the production memory T cells. Forexample, an immune response is elicited against a cancer or infectiousdisease by (a) contacting a subject with an antigenic peptideimmunization composition, (b) optionally allowing a time sufficient togenerate an initial immune response, (c) contacting the subject with anucleic acid molecule immunization composition as described herein,wherein the nucleic acid molecule of the nucleic acid moleculeimmunization composition encode one or more of the same antigenicpeptides as used in step (a).

Prior to setting forth this disclosure in more detail, it may be helpfulto an understanding thereof to provide definitions of certain terms tobe used herein. Additional definitions are set forth throughout thisdisclosure.

In the present description, any concentration range, percentage range,ratio range, or integer range includes the value of any integer withinthe recited range and, when appropriate, fractions thereof (such as onetenth or one hundredth of an integer), unless otherwise indicated. Also,any number range recited herein relating to any physical feature (suchas polymer subunits, size or thickness) include any integer within therecited range, unless otherwise indicated. As used herein, the term“about” means (1)±20% of the indicated range, value or structure; (2) avalue that includes the inherent variation of error for the method beingemployed to determine the value; or (3) a value that includes thevariation that exists among replicate experiments, unless otherwiseindicated. As used herein, the terms “a” and “an” refer to “one or more”of the enumerated components. The use of the alternative (e.g., “or”)means either one, both, or any combination thereof of the alternativesor enumerated components. As used herein, the terms “include,” “have”and “comprise” are used synonymously, which terms and variants thereofare intended to be construed as non-limiting.

The term “multiplex translation leader sequence,” “multiplex translationinitiation sequence,” or “MTI” refers to a nucleic acid moleculecomprising at least one non-conventional CUG start codon in addition tothe one standard ATG start codon. In some embodiments, an MTI allows theproduction of more than one mole of protein per mole of mRNA. In certainembodiments, an MTI nucleic acid molecule corresponds to a 5′-portion ofan FGF2 gene (e.g., a human FGF2 gene as set forth in GenBank AccessionNo. NM_002006.4) containing (a) the ATG start codon of FGF2 and (b) asequence comprising about 123 nucleotides to about 385 nucleotidesupstream (5′) of the ATG start codon of FGF2, wherein this portionupstream of the ATG start codon comprises from one to threetranslationally active non-conventional (e.g., CUG) start codons. Incertain embodiments, an MTI further comprises (c) about 15 nucleotides(encoding about 5 amino acids) to about 45 nucleotides (encoding about15 amino acids) downstream (3′) of the ATG start codon of FGF2. In someembodiments, an MTI comprises (d) at least one to two nuclearlocalization domains located upstream of the AUG start codon anddownstream of at least one non-conventional CUG start codon.

For example, a multiplex translation initiation sequence comprising fourFGF2 translation initiation sites (three non-conventional CUG startcodons and one standard ATG start codon) linked upstream of a nucleicacid molecule encoding a fusion protein (e.g., a plurality of antigenpeptides linked in a linear array) can yield four moles of amulti-peptide fusion protein translation product for every one mole oftranscribed mRNA. In certain embodiments, a nucleic acid moleculeencoding a fusion protein is introduced into a host cell and expressed,wherein the nucleic acid molecule contains a multiplex translationleader sequence comprising at least one non-AUG (e.g., CUG) translationinitiation site that mediates translation initiation at the non-AUG(e.g., CUG) translation initiation site. In some embodiments, the MTIcomprises at least five translation initiation sites (fournon-conventional CUG start codons and one standard ATG start codon).

Non-canonical translation initiation start site have been described byFlorkiewicz et al., BBRC 409(3):494-499, 2011; Ivanov et al., NucleicAcids Res 39(10), 2011; Peabody D S, J Biological Chem 264(9):5031-5035,1989; Starck et al., Science 336(6089):1719-23, 2012; Hann et al., Genesand Dev 6:1229-1240, 1992; Touriol et al., Biol Cell 95(3-4):169-78,2003; Schwab et al., Science 301(5638):1367-71, 2003; Malarkannan etal., Immunity 10(6):681-90, 1999, all of which are incorporated byreference in entirety. In certain situations, classical AUG mediatedtranslation may be inhibited while translation initiation from non-AUGinitiation codons may continue or may even be enhanced.

As used herein, “tumor associated antigen” or “TAA” refers to a protein,peptide, or variant thereof that is preferentially expressed on cancercells or pre-cancer cells that exhibit deregulated growth.“Preferentially expressed” refers to expression of detectable levels ofthe protein or peptide in a cell or on the surface of a cell, whereinthe protein or peptide is not expressed on normal cells of the sametype. Preferably, the TAA is not expressed on non-cancer cells.Exemplary, TAAs include HER2/neu, BRAF, BRCA1/2, folate receptor-α, WT1,PI3K, NY-ES01, GNRH1, CTAG1A, CEA, IGFBP2, Cyclin D1, and MIF. The terms“tumor associated antigen,” “TAA,” “biomarker,” “cancer marker,” and“marker” are used interchangeably throughout.

A “fusion protein” or “chimeric protein,” as used herein, refers to alinear single chain protein that includes polypeptide components basedon one or more parental proteins, polypeptides, or fragments thereof(e.g., antigenic peptides) and does not naturally occur in a host cell.A fusion protein can contain two or more naturally-arising amino acidsequences that are linked together in a way that does not occurnaturally. For example, a fusion protein may have two or more portionsfrom the same protein or a fragment thereof (e.g., antigenic fragment)linked in a way not normally found in a cell or a protein, or a fusionprotein may have portions (e.g., antigenic portions) from two, three,four, five or more different proteins linked in a way not normally foundin a cell. Also, a fusion protein may have two or more copies of thesame portion of a protein or a fragment thereof (e.g., antigenicfragment). A fusion protein can be encoded by a nucleic acid moleculewherein a polynucleotide sequence encoding one protein or a portionthereof (e.g., antigen) is appended in frame with a nucleic acidmolecule that encodes one or more proteins or a portion thereof (e.g.,same or different antigens), which two or more proteins or portionsthereof are optionally separated by nucleotides that encode a linker,spacer, cleavage site, junction amino acids, or a combination thereof.The valency of any one or more antigenic peptide epitope of thecompositions herein, for example of a fusion protein comprisingantigenic peptides, may be increased by duplicating, tripling,quadrupling, or further expanding the number of individual antigenicpeptide epitopes contained therein.

A “spacer” refers to an amino acid sequence that connects two proteins,polypeptides, peptides, or domains and may provide a spacer functioncompatible with cleavage of a linear antigen array into individualantigenic peptides capable of associating with an MHC (HLA) molecule. Aspacer can promote proteolytic processing into antigenic peptides byenhancing or promoting a disordered conformation of a primarytranslation product and thereby promoting its ubiquitin modification.

“Junction amino acids” or “junction amino acid residues” refer to one ormore (e.g., about 2-10) amino acid residues between two adjacent motifs,regions or domains of a polypeptide, such as between a antigenicpeptides or between an antigenic peptide and an adjacent peptide encodedby a multiplex translation leader sequence or between an antigenicpeptide and a spacer or cleavage site. Junction amino acids may resultfrom the construct design of a fusion protein (e.g., amino acid residuesresulting from the use of a restriction enzyme site during theconstruction of a nucleic acid molecule encoding a fusion protein).Junction amino acids may be derived from a sequence authentic or nativeto a T-cell antigenic epitope sequence by extending the NH2 and/orCOOH-terminus of a HLA Class 1 or HLA Class II peptide identified bycomputer algorithm or other means such as mass spectrometry analysis ofpeptides eluted from HLA Class I or HLA Class II restricted proteincomplexes.

As used herein, a molecule or compound “consists essentially of” one ormore domains or encodes one or more domains “consisting essentially of”(e.g., an antigen, a linker or spacer, a proteolytic cleavage site, anuclear localization signal, a multiplex translation initiationsequence) when the portions outside of the one or more domains (e.g.,amino acids at the amino- or carboxy-terminus or between domains), incombination, contribute to no more than 20% (e.g., no more than 15%,10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of the molecule orcompound and do not substantially affect (i.e., do not alter theactivity by more than 50% (e.g., no more than 40%, 30%, 25%, 20%, 15%,10%, 5%, 4%, 3%, 2%, 1%) of the activities of one or more of the variousdomains (e.g., the immunogenicity of an antigen or antigenic epitope,the capability of localizing to the nucleus, the capability of forming apolypeptide complex (such as an HLA-peptide complex), the capability ofpromoting translation initiation from non-AUG codons). In certainembodiments, a nucleic acid molecule consists essentially of a multiplextranslation leader sequence and a sequence encoding one or moreantigens, antigenic epitopes or combination thereof, wherein an encodedantigen may comprise junction amino acids at the amino- and/orcarboxy-terminus or between antigens. In certain embodiments, suchjunction amino acids between antigens or antigenic epitopes formproteolytic cleavage sites such that the antigens or antigenic epitopesare separated in vivo and can associate, for example, with acorresponding mammalian class I or class II HLA or MHC molecule.

As used herein, “nucleic acid” or “nucleic acid molecule” refers to anyof deoxyribonucleic acid (DNA), ribonucleic acid (RNA),oligonucleotides, fragments generated, for example, by the polymerasechain reaction (PCR) or by in vitro translation, and fragments generatedby any one or more of ligation, scission, endonuclease action, orexonuclease action. In certain embodiments, the nucleic acids of thepresent disclosure are produced by PCR. Nucleic acids may be composed ofmonomers that are naturally occurring nucleotides (such asdeoxyribonucleotides and ribonucleotides), analogs of naturallyoccurring nucleotides (e.g., α-enantiomeric forms of naturally-occurringnucleotides), or a combination thereof. Modified nucleotides can havemodifications in or replacement of sugar moieties, or pyrimidine orpurine base moieties. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, morpholino, or the like. The term“nucleic acid molecule” also includes “peptide nucleic acids” (PNAs),which comprise naturally occurring or modified nucleic acid basesattached to a polyamide backbone. Nucleic acid molecules can be eithersingle stranded or double stranded.

The term “construct” refers to any polynucleotide that contains arecombinant nucleic acid. A construct may be present in a vector (e.g.,a bacterial vector, a viral vector) or may be integrated into a genome.A “vector” is a nucleic acid molecule that is capable of transportinganother nucleic acid. Vectors may be, for example, plasmids, cosmids,viruses, a RNA vector, or a linear or circular DNA or RNA molecule thatmay include chromosomal, non-chromosomal, semi-synthetic or syntheticnucleic acids. Exemplary vectors are those capable of autonomousreplication (episomal vector) and/or expression of nucleic acids towhich they are linked (expression vectors).

Viral vectors include retrovirus, adenovirus, parvovirus (e.g.,adeno-associated viruses), coronavirus, negative strand RNA viruses suchas ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabiesand vesicular stomatitis virus), paramyxovirus (e.g., measles andSendai), positive strand RNA viruses such as picornavirus andalphavirus, and double-stranded DNA viruses including adenovirus,herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barrvirus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox andcanarypox). Other viruses include Norwalk virus, togavirus, flavivirus,reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.Examples of retroviruses include avian leukosis-sarcoma, mammalianC-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus,spumavirus (Coffin, J. M., Retroviridae: The viruses and theirreplication, In Fundamental Virology, Third Edition, B. N. Fields, etal., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

“Lentiviral vector,” as used herein, means HIV-based lentiviral vectorsthat are useful for gene delivery because of their relatively largepackaging capacity, reduced immunogenicity and their ability to stablytransduce with high efficiency a large range of different cell types.Lentiviral vectors are usually generated following transienttransfection of three or more plasmids (e.g., packaging, envelope, andtransfer) into producer cells. Like HIV, lentiviral vectors enter thetarget cell through the interaction of viral surface glycoproteins withreceptors on the cell surface. On entry, the viral RNA undergoes reversetranscription, which is mediated by the viral reverse transcriptasecomplex. The product of reverse transcription is a double-strandedlinear viral DNA, which is the substrate for viral integration in theDNA of infected cells.

The term “operably-linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably-linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., the coding sequence is under thetranscriptional control of the promoter). “Unlinked” means that theassociated genetic elements are not closely associated with one anotherand the function of one does not affect the other.

As used herein, “expression vector” refers to a DNA construct containinga nucleic acid molecule that is operably-linked to a suitable controlsequence capable of effecting the expression of the nucleic acidmolecule in a suitable host. Such control sequences include a promoterto effect transcription, an optional operator sequence to control suchtranscription, a sequence encoding suitable mRNA ribosome binding sites,and sequences which control termination of transcription andtranslation. The vector may be a plasmid, a phage particle, a virus, orsimply a potential genomic insert. A viral vector may be DNA (e.g., anAdenovirus or Vaccinia virus) or RNA-based including an oncolytic virusvector (e.g., VSV), replication competent or incompetent. Oncetransformed into a suitable host, the vector may replicate and functionindependently of the host genome, or may, in some instances, integrateinto the genome itself. In the present specification, “plasmid,”“expression plasmid,” “virus” and “vector” are often usedinterchangeably.

The term “expression,” as used herein, refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation. Translation mayinitiate from a non-conventional start codon, such as a CUG codon, ortranslation may initiate from several start codons (standard AUG andnon-conventional) to produce more protein (on a per mole amount) thanmRNA produced.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell wherein the nucleicacid sequence may be incorporated into the genome of the cell (e.g.,chromosome, plasmid, plastid, or mitochondrial DNA), converted into anautonomous replicon, or transiently expressed (e.g., transfected mRNA).

As used herein, “contacting,” “contacting a cell,” “contacting asubject” or variants thereof, in the context of contacting with anucleic acid molecule or composition refers to introducing the nucleicacid into the cell such that the at least some of the encoded content ofthe nucleic acid molecule is expressed in the cell. By “introduced” ismeant transformation, transfection, transduction, or other methods knownin the art for the introduction of nucleic acid molecules such as by agene gun or nanoparticles.

Expression of recombinant proteins may be inefficient outside theiroriginal host since codon usage bias has been observed across differentspecies of bacteria (Sharp et al., Nucl. Acids Res. 33:1141, 2005). Evenover-expression of recombinant proteins within their native host may bedifficult. In certain embodiments, nucleic acid molecules (e.g., nucleicacids encoding antigenic peptides) to be introduced into a host asdescribed herein may be subjected to codon optimization prior tointroduction into the host to ensure protein expression is enhanced.“Codon optimization” refers to the alteration of codons in genes orcoding regions of nucleic acids before transformation to reflect thetypical codon usage of the host without altering the polypeptide encodedby the DNA molecule. Codon optimization methods for optimum geneexpression in heterologous hosts have been previously described (see,e.g., Welch et al., PLoS One 4:e7002, 2009; Gustafsson et al., TrendsBiotechnol. 22:346, 2004; Wu et al., Nucl. Acids Res. 35:D76, 2007;Villalobos et al., BMC Bioinformatics 7:285, 2006; U.S. PatentPublication Nos. 2011/0111413 and 2008/0292918; disclosure of which areincorporated herein by reference, in their entirety). In certainembodiments, the multiplex translation leader sequence of thisdisclosure is human and is not codon optimized. Codon optimizedrecombinant nucleic acids may be distinguished from correspondingendogenous genes based on the use of PCR primers designed to recognize acodon optimized portion that is consequently distinct from a non-codonoptimized portion of a nucleic acid.

The terms “identical” or “percent identity,” in the context of two ormore polypeptide or nucleic acid molecule sequences, means two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same overa specified region (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity), when compared andaligned for maximum correspondence over a comparison window, ordesignated region, as measured using methods known in the art, such as asequence comparison algorithm, by manual alignment, or by visualinspection. For example, a preferred algorithm suitable for determiningpercent sequence identity and sequence similarity is the BLAST 2.0algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403,1990, with the parameters set to default values.

As used herein, TAP (Transporter Associated with Antigen Processing),refers to a heterodimer comprising a TAP1 and a TAP2 protein.Heterodimers of TAP are located in the endoplasmic reticulum (ER) whereit functions to move selected peptides from the cytosol into the lumenof the ER where the peptide binds to a HLA Class I protein. Anotherintracellular TAP protein is TAPL (transporter associated with antigenprocessing like protein) localized to intracellular vesicularcompartments such as the endosome and lysosome. TAP1, TAP2, and TAPL aremembers of the ATP binding cassette (ABC) transporter family. However,unlike ER localized TAP1/2 heterodimers, TAPL functions as a homodimer.Examples of TAP include TAP1 (GenBank No. NM_000593.5), TAP2 (GenBankNo. NM_000544.3), and TAPL (GenBank No. NM_019624.3).

As used herein, “mutation” refers to a change in the sequence of anucleic acid molecule or polypeptide molecule as compared to a referenceor wild-type nucleic acid molecule or polypeptide molecule,respectively. A mutation can result in several different types ofchanges in sequence, including substitution, insertion or deletion ofnucleotide(s) or amino acid(s). In other embodiments, a mutation is asubstitution of one or more nucleotides or residues. In certainembodiments, an altered or mutated protein or polypeptide only containsconservative amino acid substitutions as compared to the referencemolecule. In certain other embodiments, an altered or mutated protein orpolypeptide only contains non-conservative amino acid substitutions ascompared to the reference molecule. In yet other embodiments, an alteredor mutated protein or polypeptide contains both conservative andnon-conservative amino acid substitutions. In any of these embodiments,an alteration or mutation does not alter or eliminate an antigenicepitope of a protein or peptide and the altered or mutated peptide isstill recognized by its cognate MEW (HLA) molecule.

A “conservative substitution” is recognized in the art as a substitutionof one amino acid for another amino acid that has similar properties.Exemplary conservative substitutions are well known in the art (see,e.g., WO 97/09433, page 10, published Mar. 13, 1997; Lehninger,Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp.71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press,Cambridge, Mass. (1990), p. 8). In certain embodiments, a conservativesubstitution includes, for example, a leucine to serine substitution.

As used herein, “recombinant” or “non-natural” refers to an organism,microorganism, cell, nucleic acid molecule, or vector that has at leastone engineered genetic alteration or has been modified by theintroduction of a heterologous nucleic acid molecule, or refers to acell that has been altered such that the expression of an endogenousnucleic acid molecule or gene can be controlled. Recombinant also refersto a cell that is derived from a non-natural cell or is progeny of anon-natural cell having one or more such modifications. Geneticalterations include, for example, modifications introducing expressiblenucleic acid molecules encoding proteins, or other nucleic acid moleculeadditions, deletions, substitutions or other functional alteration of acell's genetic material. For example, recombinant cells may expressgenes or other nucleic acid molecules that are not found in identical orhomologous form within a native (wild-type) cell (e.g., a fusion orchimeric protein), or may provide an altered expression pattern ofendogenous genes, such as being over-expressed, under-expressed,minimally expressed, or not expressed at all.

Recombinant methods for expression of exogenous or heterologous nucleicacids in cells are well known in the art. Such methods can be founddescribed in, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York(2001); and Ausubel et al., Current Protocols in Molecular Biology, JohnWiley and Sons, Baltimore, Md. (1999). Exemplary exogenous proteins orenzymes to be expressed include TAP1, TAP2, antigens, cytokines, or anycombination thereof. Genetic modifications to nucleic acid moleculesencoding fusion proteins can confer a biochemical or metaboliccapability to a recombinant or non-natural cell that is altered from itsnaturally occurring state.

As used herein, the term “endogenous” or “native” refers to a gene,protein, compound or activity that is normally present in a host cell.The term “homologous” or “homolog” refers to a molecule or activity froman exogenous (non-native) source that is the same or similar molecule oractivity as that found in or derived from a host or host cell.

As used herein, “heterologous” nucleic acid molecule, construct orsequence refers to a nucleic acid molecule or portion of a nucleic acidmolecule sequence that is not native to a cell in which it is expressed,a nucleic acid molecule or portion of a nucleic acid molecule native toa host cell that has been altered or mutated, or a nucleic acid moleculewith an altered expression as compared to the native expression levelsunder similar conditions. For example, a heterologous control sequence(e.g., promoter, enhancer) may be used to regulate expression of a geneor a nucleic acid molecule in a way that is different than the gene or anucleic acid molecule that is normally expressed in nature or culture.In certain embodiments, a heterologous nucleic acid molecule may behomologous to a native host cell gene, but may have an alteredexpression level or have a different sequence or both. In otherembodiments, heterologous or exogenous nucleic acid molecules may not beendogenous to a host cell or host genome (e.g., fusion protein), butinstead may have been introduced into a host cell by transformation(e.g., transfection, electroporation), wherein the added molecule mayintegrate into the host genome or can exist as extra-chromosomal geneticmaterial either transiently (e.g., mRNA) or semi-stably for more thanone generation (e.g., episomal viral vector, plasmid or otherself-replicating vector).

In certain embodiments, more than one heterologous or exogenous nucleicacid molecule can be introduced into a host cell as separate nucleicacid molecules, as a polycistronic nucleic acid molecule, as a singlenucleic acid molecule encoding a fusion protein, or any combinationthereof, and still be considered as more than one heterologous orexogenous nucleic acid. When two or more exogenous nucleic acidmolecules are introduced into a host cell, it is understood that the twomore exogenous nucleic acid molecules can be introduced as a singlenucleic acid molecule (e.g., on a single vector), on separate vectors,as single or multiple mRNA molecules, integrated into the hostchromosome at a single site or multiple sites, and each of theseembodiments is still to be considered two or more exogenous nucleic acidmolecules. Thus, the number of referenced heterologous nucleic acidmolecules or protein activities refers to the number of encoding nucleicacid molecules or the number of protein activities, not the number ofseparate nucleic acid molecules introduced into a host cell.

For example, a cell can be modified to express two or more heterologousor exogenous nucleic acid molecules, which may be the same or different,that encode one or more fusion proteins, as disclosed herein. In certainembodiments, a host cell will contain a first nucleic acid moleculeencoding a first fusion protein and a separate second nucleic acidmolecule encoding a second fusion protein, or a host cell will contain asingle polycistronic nucleic acid molecule that encodes a first fusionprotein and second fusion protein, or single nucleic acid molecule thatencodes a first fusion protein, a cleavable amino acid sequence (e.g.,trypsin, pepsin, proteasome site) or a self-cleaving amino acid sequence(e.g., 2A protein), and a second fusion protein.

“T cell receptor” (TCR) is a molecule found on the surface of T cellsthat, along with CD3, is generally responsible for recognizing antigensbound to major histocompatibility complex (WIC) molecules. A TCRconsists of a disulfide-linked heterodimer of the highly variable α andβ chains in most T cells. In other T cells, an alternative receptor madeup of variable γ and δ chains is expressed. Each chain of the TCR is amember of the immunoglobulin superfamily and possesses oneamino-terminal immunoglobulin variable domain, one immunoglobulinconstant domain, a transmembrane region, and a short cytoplasmic tail atthe C-terminal end (see, Abbas and Lichtman, Cellular and MolecularImmunology (5th Ed.), Editor: Saunders, Philadelphia, 2003; Janeway etal., Immunobiology: The Immune System in Health and Disease, 4^(th) Ed.,Current Biology Publications, p148, 149, and 172, 1999).

HLA Class I binding CD8+ T-cell are T-cells that kill tumor cells orvirally infected cells through interactions between an antigenic peptidebound cell surface localized HLA (also known as MHC). Cells that presentHLA class I antigenic peptide epitopes are referred to as antigenpresenting cells.

HLA Class II presentation is different than HLA Class I in that peptideantigens are bound to HLA class II complexes that are already on thecell surface or that are present in certain intracellular vesicles, suchas endosomes or lysosomes. Class II antigen presentation thus does not apriori require the activity of TAP. However, the protein referred to asTAPL, which is found localized to certain intracellular vesicles such asan endosome or lysosome, may function to recognize cytosol localizedclass II peptides and consequently transfer a 12-18 amino acid peptidefrom the cytosol into a intracellular vesicle containing a Class II HLAthat then traffics back onto the cell surface where it can then interactwith and consequently expand a population of CD4+ T-cells. A host'sT-cells that recognized cell surface localized HLA class II peptidecomplexes are called CD4+ T-cells, which are principally responsible forpreserving the function of CD8+ killer T-cells mediated by the releaseof a set of CD8+ cytokines.

The term “antigen specific T-cell response” refers to an immune responsemediated by T-cells directed at a cell expressing a specific antigen. Insome embodiments, the T-cell response is a CD8+ T-cell response, a CD4+T-cell response, or a combination thereof.

The term “antigen, “antigenic peptide” or variants thereof refers to apolypeptide that can stimulate a cellular immune response. In someembodiments, an antigen is an HLA Class I, an HLA Class II peptide, orHLA Class II peptide having an embedded HLA Class I peptide.

The term “immunization composition” refers to a composition that canstimulate or elicit an immune response. Preferably, the immune responseis a cellular immune response, such as an adaptive immune responsemediated by T-cells (e.g., CD8+ T-cells or CD4+ T-cells). In someembodiments, an immunization composition is a pharmaceuticalformulation. In further embodiments, an immunization composition is anantigenic peptide immunization composition, a nucleic acid immunizationcomposition, a cell immunization composition, or a combination thereof.

The term “antigen immunization composition” or “peptide immunizationcomposition” refers to an immunization composition that includes one ormore antigens that are capable of promoting or stimulating a cellularimmune response. In some embodiments, an antigen immunizationcomposition comprises an HLA Class I peptide, HLA Class II peptide, HLAClass II peptide having an embedded HLA Class I peptide, or combinationsthereof.

The term “nucleic acid immunization composition” refers to animmunization composition that includes a nucleic acid molecule thatencodes one or more antigens or antigenic epitopes, and that can becontained in a vector (e.g., plasmid, virus). A nucleic acidimmunization composition can be introduced into a host cell ex vivo, orin vivo for expression of the one or more antigenic peptides in asubject. In certain embodiments, a nucleic acid immunization compositionencodes an HLA Class I peptide, HLA Class II peptide, HLA Class IIpeptide having an embedded HLA Class I peptide, or combinations thereof.

The term “nucleic acid molecule immunization” or “DNA immunization” asused herein refers to a nucleic acid molecule encoding one or moreantigens introduced into a host or host cell in order to express the oneor more antigens in vivo. A nucleic acid molecule immunization can be bydirect administration into a host, such as by standard injection (e.g.,intramuscular, intradermal), transdermal particle delivery, inhalation,topically, orally, intranasally, or mucosally. Alternatively, a nucleicacid molecule can be introduced ex vivo into host cells (e.g., hostcells or cells from a donor HLA matched to the host) and the transfectedhost cells can be administered into the host such that an immuneresponse can be mounted against the one or more antigens encoded by thenucleic acid molecule.

The term “nucleic acid molecule vaccine” or “DNA vaccine” as used hereinrefers to a nucleic acid molecule encoding one or more antigens orantigenic epitopes that is used in a nucleic acid molecule immunizationas defined herein.

“Treatment,” “treating” or “ameliorating” refers to medical managementof a disease, disorder, or condition of a subject (e.g., patient), whichmay be therapeutic, prophylactic/preventative, or a combinationtreatment thereof. A treatment may improve or decrease the severity atleast one symptom of a disease, delay worsening or progression of adisease, or delay or prevent onset of additional associated diseases.“Reducing the risk of developing a disease” refers to preventing ordelaying onset of a disease (e.g., cancer) or reoccurrence of one ormore symptoms of the disease.

A “therapeutically effective amount (or dose)” or “effective amount (ordose)” of a compound or composition refers to that amount of compoundsufficient to result in amelioration of one or more symptoms of thedisease being treated in a statistically significant manner. The preciseamount will depend upon numerous factors, e.g., the activity of thecomposition, the method of delivery employed, the immune stimulatingability of the composition, the intended patient and patientconsiderations, or the like, and can readily be determined by one ofordinary skill in the art.

A therapeutic effect may include, directly or indirectly, the reductionof one or more symptoms of a disease (e.g., reduction in tumor burden orreduction in pathogen load). A therapeutic effect may also include,directly or indirectly, the stimulation of a cellular immune response.

A “matched” vaccination strategy is one in which a peptide vaccine and aDNA vaccine are administered to a subject wherein the peptides of thepeptide vaccine and peptides encoded by the DNA vaccine are derived fromthe same protein (e.g., same cancer related TAA or pathogen derivedantigen). In some embodiments, the peptide vaccine peptides are HLAclass II antigens. In some embodiments, the peptides encoded by the DNAvaccine are HLA class I peptides. A matched vaccination strategy caninclude administering respective compositions as a prime-and-boost.

As used herein, “prime-and-boost” or “immunogenic challenge” refers tosequentially or simultaneously delivering one or more of a series ofpeptide vaccines followed by one or more in a series of DNA vaccines,or, in the alternative, sequentially or simultaneously delivering one ormore of a series of DNA vaccines followed by one or more in a series ofpeptide vaccines.

Cancer cells may aberrantly express certain polypeptides, either byinappropriate expression or overexpression. As such, inappropriatelyexpressed polypeptides have been identified as tumor associated antigens(TAAs). Tumor-associated antigens however may be functionallynon-immunogenic or are ineffectively or weakly immunogenic. This may bereferred to immune tolerance. Compositions and methods of the instantdisclosure are designed to enhance or further stimulate a patient'simmune system so that it is capable of functioning more effectively tokill cancer cells or to kill pathogen infected cells.

As used herein, a “subject,” may be any organism capable of developing acellular immune response, such as humans, pets, livestock, show animals,zoo specimens, or other animals. For example, a subject may be a human,a non-human primate, dog, cat, rabbit, rat, mouse, guinea pig, horse,cow, sheep, goat, pig, or the like. Subjects in need of administrationof therapeutic agents as described herein include subjects at high riskfor developing a cancer or infectious disease as well as subjectspresenting with an existing cancer or infectious disease. A subject maybe at high risk for developing a cancer if the subject has experiencedan injury, such as exposure to carcinogens or radiation, or has agenetic predisposition, such as a mutation in the BRCA1/2, folatereceptor-α, or p53 genes. Subjects suffering from or suspected of havingan infectious disease or a cancer can be identified using methods asdescribed herein and known in the art.

A “subject in need” refers to a subject at high risk of, or sufferingfrom, a disease, disorder or condition that is amenable to treatment oramelioration with a compound or a composition thereof provided herein.In certain embodiments, a subject in need is a human.

Accordingly, in some embodiments the composition of the instantdisclosure provides a chimeric nucleic acid molecule, comprising amultiplex translation initiation (MTI) sequence and a nucleic acidmolecule encoding an antigen, an antigenic epitope, or a combinationthereof, wherein the MTI comprises at least one non-AUG translationinitiation site that mediates translation initiation of the antigen,antigenic epitope, or combination thereof.

A nucleic acid based plasmid or virus delivery system as describedherein provides a transcribed mRNA that is recognized by eukaryotic celltranslation components, which is translated into a protein following theinitiation of translation that occurs at the first or appropriatetranslation initiation codon. The canonical mechanism of translationinitiation starts at an AUG codon. However, as used herein, translationinitiation can also begin at a non-AUG codon, such as a CUG codon. Thehuman gene encoding FGF2 is an example of a gene with translation thatis mediated via three CUG start codons, in addition to one AUG codon. Insome embodiments, the MTI nucleic acid molecule set forth hereincomprises a portion of the FGF2 multiple translation initiation domainof the gene/mRNA.

In some embodiments, an MTI of the chimeric nucleic acid moleculecorresponds to a 5′-portion of an FGF2 gene (e.g., a human FGF2 gene asset forth in GenBank Accession No. NM_002006.4) containing the ATG startcodon of FGF2 and a sequence comprising about 123 nucleotides to about385 nucleotides upstream (5′) of the ATG start codon of FGF2, whereinthis portion upstream of the ATG start codon comprises from one to threetranslationally active non-conventional (e.g., CUG) start codons. Incertain embodiments, the MTI further comprises about 15 nucleotides(encoding about 5 amino acids) to about 45 nucleotides (encoding about15 amino acids) downstream (3′) of the ATG start codon of FGF2. In someembodiments, the MTI comprises at least one to two nuclear localizationdomains located upstream of the AUG start codon and downstream of atleast one non-conventional CUG start codon. In certain embodiments, anMTI sequence comprising four FGF2 translation initiation sites (threenon-conventional CUG start codons and one standard ATG start codon)linked upstream of a nucleic acid molecule encoding a fusion protein(e.g., a plurality of antigen peptides linked in a linear array) canyield four moles of a multi-peptide fusion protein translation productfor every one mole of transcribed mRNA. In certain embodiments, anucleic acid molecule encoding a fusion protein is introduced into ahost cell and expressed, wherein the nucleic acid molecule contains amultiplex translation leader sequence comprising at least one non-AUG(e.g., CUG) translation initiation site that mediates translationinitiation at the non-AUG (e.g., CUG) translation initiation site. Insome embodiments, an MTI sequence is a nucleic acid molecule having asequence as set forth in any one of SEQ ID NOS.: 1-6, 95, or 96. Incertain embodiments, an MTI sequence is nucleic acid molecule having atleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity to the sequence set forth in any one of SEQ IDNOS.:1-6.

In certain embodiments, a nucleic acid molecule encoding a fusionprotein comprises a plurality of class I MHC (HLA) antigenic peptides, aplurality of class II MHC (HLA) antigenic peptides, or a combinationthereof. In further embodiments, a plurality of MHC (HLA) class Iantigenic peptides, class II antigenic peptides, or a combinationthereof are expressed as a linear single-chain polypeptide antigenarray, wherein each antigen is separated from the adjacent antigen by aspacer, a cleavable site (e.g., enzyme recognition site orself-cleaving), or both, and wherein the polypeptide antigen array isprocessed by the cellular machinery (e.g., proteasome or nuclearproteasome) into individual antigen peptides capable of forming acomplex with their cognate MHC molecules.

In accordance with the disclosure set forth herein, a translatedpolypeptide or protein comprises, for example, a linear peptide antigenarray (PAA) that is or may then be modified intracellularly by theaddition of a ubiquitin moiety. The ubiquitin-modified PAA is recognizedby and proteolytically processed through the actions of the proteasome.The results of proteolytic processing are small peptides approximately8-12 amino acids in length that are then recognized by the intracellularER localized protein termed TAP.

Accordingly, in some embodiments, the chimeric nucleic acid moleculeincludes a nucleic acid molecule that encodes one or more antigens,antigenic epitopes, or a combination thereof (referred to collectivelyas antigens), which is referred to herein as a polyantigen array (PAA).In some embodiments, a PAA encodes one or more antigens from the sameprotein. In other embodiments, a PAA encodes one or more antigens frommore than one protein (i.e., comprises a chimeric polypeptide). Forexample, a PAA can encode one or more antigens from folate receptor-αand one or more antigens from Her2/neu. In some embodiments, a nucleicacid molecule encodes a plurality of antigens ranging from about 2 toabout 20, from about 2 to about 15, from about 2 to about 10, from about2 to about 9, from about 2 to about 8, or a nucleic acid moleculeencodes 2, 4, 5, 6, 7, or 8 antigens.

In some embodiments, a PAA encodes a plurality of antigens wherein twoor more of the plurality of antigens are separated by a spacer. Incertain embodiments, a spacer is comprised of about 2 to about 35 aminoacids, or about 5 to about 25 amino acids or about 8 to about 20 aminoacids or about 10 to about 15 amino acids. In some embodiments, a spacermay have a particular sequence, such as a (G₄S)_(n) repeat, wherein n isan integer from 1-20, from 1-15, from 1-10, from 1-5, from 1-3, or thelike. In particular embodiments, a spacer is a (G₄S)₂ peptide.

In some embodiments, a chimeric nucleic acid encodes a PAA wherein twoor more of the plurality of antigens are separated by a cleavage site.In certain embodiments, a cleavage site comprises from about 2 to about20 amino acids amino-terminal to the antigenic peptide as found in thereference protein, from about 2 to about 20 amino acids carboxy-terminalto the antigenic peptide as found in the reference protein, aself-cleaving amino acid sequence, or a combination thereof. In certainembodiments, the cleavage site comprises from about 2 to about 15, about2 to about 10, or about 2 to about 5 amino acids at the amino-terminalor the carboxy-terminal end of the antigen. In some embodiments, thecleavage site is a self-cleaving amino acid sequence comprising a 2Apeptide from porcine teschovirus-1 (P2A), equine rhinitis A virus (E2A),Thosea asigna virus (T2A), foot-and-mouth disease virus (F2A), or anycombination thereof (see, e.g., Kim et al., PLOS One 6:e18556, 2011,which 2A nucleic acid and amino acid sequences are incorporated hereinby reference in their entirety).

In some embodiments, an antigen or antigenic epitope is an HLA Class Iantigenic peptide, an HLA Class II antigenic peptide, an HLA class IIantigenic peptide with an embedded HLA Class I antigenic peptide, or anycombination thereof. In certain embodiments, an antigen or antigenicepitope is an HLA class II antigenic peptide comprising an embedded HLAClass I antigenic peptide. An “embedded antigen” is an antigenicsequence or epitope that is contained within a larger antigenic sequenceor epitope. For example, a sequence corresponding to an antigenic HLAClass II peptide antigen may contain within it a sequence representativeof an HLA Class I antigenic peptide. In some embodiments, an extendedantigenic peptide sequence may contain multiple overlapping (i.e.,embedded) antigens.

Cancer cells may be distinguished from normal cells by the de novoexpression of one or more marker proteins or tumor-associated antigens(TAA). A marker protein or a TAA may comprise one or more antigenicpeptides. For example, antigenic peptides may represent either HLA ClassI or HLA Class II restricted antigenic peptide epitopes. A TAA or cancermarker protein or antigenic peptides thereof may be used in a vaccinecomposition capable of eliciting an immune response (e.g., a cellularimmune response, such as an antigen-specific T cell response) targetingthe unwanted cancer cell for destruction by the patients' immune system.

In some embodiments, an antigen or antigenic epitope is atumor-associated antigen (TAA). An antigenic peptide of a PAA disclosedherein may be derived from one or more TAAs. A TAA can be an antigenassociated with breast cancer, triple negative breast cancer,inflammatory breast cancer, ovarian cancer, uterine cancer, colorectalcancer, colon cancer, primary peritoneal cancer, testicular cancer,renal cancer, melanoma, glioblastoma, lung cancer, or prostate cancer.In certain embodiments, a TAA derived antigenic T-cell epitope (eitherClass I or Class II) is from a HER2/neu, folate receptor alpha, CyclinD1, IGFBP2, macrophage migration inhibitory factor (MIF), humancarcinoembryonic antigen (CEA), gonadotropin releasing hormone (GnRH),melanoma related gp100 as well as MAGE-2 and MAGE-3, a testis cancerantigen (e.g., NY-ESO-1), cancer/testis antigen 1A (CTAG1A), Wilms tumorprotein 1 (WT1), p53, BRCA1, BRCA2, PI3K, BRAF, insulin-like growthfactor binding protein 2, or PD-1 antagonists. In some embodiments, aPAA as used herein is a combination of T-cell antigenic epitopes frommore than one TAA (e.g., a combination of a HER2/neu and folate receptoralpha antigenic T-cell epitopes). In certain embodiments, a TAAcomprises a HER2/neu antigen, a folate receptor-α antigen, or acombination thereof. Exemplary folate receptor-α antigenic peptides arepresented in U.S. Pat. No. 8,486,412, which peptides are hereinincorporated by reference. Exemplary HER2/neu, Cyclin D1, IGFBP2, andCEA antigenic peptides are described in Table I and II of U.S.Application Publication No. US 2010/0310640, which antigenic peptidesare herein incorporated by reference in their entirety.

In certain embodiments, a TAA has an amino acid sequence derived fromHER2 as set forth in SEQ ID NOS.:117-135, or any combination thereof. Insome embodiments, the TAA has an amino acid sequence derived from folatereceptor-α as set forth in SEQ ID NOS.:69-93, or any combinationthereof. Accordingly, in some embodiments, the TAA has an amino acidsequence as set forth in any one of SEQ ID NOS.:69-93 or 117-135, or anycombination thereof. In certain embodiments, the nucleic acid moleculehas at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity to any one of SEQ ID NOS.:97, 98, 136, or anycombination thereof. In certain embodiments, the nucleic acid moleculeencodes an antigen having an amino acid sequence as set forth in any oneof SEQ ID NOS.:67, 68, 115, 116, or any combination thereof. In someembodiments, the acid molecule has at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNOS.:52, 57, 58, 100, 137, 139, 141, 145, or 149. In some embodiments,the TAA has an amino acid sequence derived from IGFB2 as set forth inSEQ ID NOS.:103-112, or any combination thereof. In some embodiments,the TAA has an amino acid sequence derived from CEA as set forth in SEQID NOS.:186-202, or any combination thereof. In some embodiments, theTAA has an amino acid sequence derived from Cyclin D1 as set forth inSEQ ID NOS.:152-183, or any combination thereof. In certain embodiments,the TAA has an amino acid sequence having at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to thesequence set forth in any one of SEQ ID NOS.:69-93, 103-112, 117-135,152-183, 186-202, or any combination thereof. As noted herein, anantigenic T-cell epitope may be identified using a patient's sample.

In certain embodiments, the instant disclosure provides a chimericnucleic acid molecule, comprising a multiplex translation initiation(MTI) sequence and a nucleic acid molecule encoding a fusion proteincomprising from two to about ten human folate receptor-alpha (FRα)antigenic peptides, wherein the MTI comprises at least one non-AUGtranslation initiation site that mediates translation initiation of thefusion protein and allows the production of more than one mole of fusionprotein per mole of mRNA. In some embodiments, the MTI sequence has atleast 90% sequence identity to a nucleotide sequence as set forth in anyone of SEQ ID NOS.:1-6, 95, or 96. In further embodiments, the fusionprotein comprises from two to about five antigenic peptides or comprisesfive antigenic peptides. In further embodiments, two or more of the FRαantigenic peptides of the fusion protein are separated by a spacercomprising a (G₄S)_(n) wherein n is an integer from 1 to 5. In furtherembodiments, two or more of the FRα antigenic peptides of the fusionprotein are separated by a natural cleavage site comprising from abouttwo to about ten amino acids, a self-cleaving amino acid sequence, orcombinations thereof. In further embodiments, each FRα antigenic peptideof the fusion protein has at least 90% sequence identity to any one ofSEQ ID NOS.:69-93. In further embodiments, the encoded fusion proteincomprises a polypeptide having at least 90% sequence identity with anyone of the polypeptides set forth in SEQ ID NOS.:49, 67, or 68. Infurther embodiments, the encoded fusion protein comprises a polypeptidehaving at least 90% sequence identity with any one of the polypeptidesset forth in SEQ ID NOS.:53-55 or 59-61. In further embodiments, one ormore of the FRα antigenic peptides are an HLA Class I antigenic peptide,an HLA Class II antigenic peptide, an HLA Class II antigenic peptidecomprising an embedded HLA Class I antigenic peptide, or any combinationthereof.

In certain aspects, this disclosure provides a method of eliciting acellular immune response, comprising administering to a human subject atherapeutically effective amount of a chimeric nucleic acid molecule,wherein the chimeric nucleic acid molecule comprises a multiplextranslation initiation (MTI) sequence and a nucleic acid moleculeencoding a fusion protein comprising from two to about ten human folatereceptor-alpha (FRα) antigenic peptides, wherein the MTI comprises atleast one non-AUG translation initiation site that mediates translationinitiation of the fusion protein and allows the production of more thanone mole of fusion protein per mole of mRNA, thereby eliciting acellular immune response. In further embodiments, the MTI sequence hasat least 90% sequence identity to a nucleotide sequence as set forth inany one of SEQ ID NOS.:1-6, 95, or 96. In further embodiments, each FRαantigenic peptide of the fusion protein has at least 90% sequenceidentity to any one of SEQ ID NOS.: 69-93. In further embodiments, theencoded fusion protein comprises a polypeptide having at least 90%sequence identity with any one of the polypeptides set forth in SEQ IDNOS.:49, 67, or 68. In further embodiments, the encoded fusion proteincomprises a polypeptide having at least 90% sequence identity with anyone of the polypeptides set forth in SEQ ID NOS.:53-55 or 59-61. Infurther embodiments, the chimeric nucleic acid molecule is formulated asa composition comprising a therapeutically acceptable carrier orexcipient. In further embodiments, the method comprises contacting thechimeric nucleic acid molecule with an immune cell ex vivo beforeadministration, and administering to the human subject a population ofimmune cells containing the chimeric nucleic acid molecule. In furtherembodiments, the elicited immune response treats FRα-associated cancer.

In other aspects, this disclosure provides a method of eliciting acellular immune response, comprising (a) administering to a humansubject an effective amount of an antigenic peptide immunizationcomposition comprising at least one FRα antigenic peptide, and (b)administering to the human subject an effective amount of a chimericnucleic acid molecule, wherein the chimeric nucleic acid moleculecomprises a multiplex translation initiation (MTI) sequence and anucleic acid molecule encoding a fusion protein comprising from two toabout ten human folate receptor-alpha (FRα) antigenic peptides, whereinthe MTI comprises at least one non-AUG translation initiation site thatmediates translation initiation of the fusion protein and allows theproduction of more than one mole of fusion protein per mole of mRNA,thereby eliciting a cellular immune response. In further embodiments,the chimeric nucleic acid molecule encodes one or more of the sameantigenic peptides as used in step (a). In further embodiments, step (b)is performed simultaneously with step (a), or step (b) is performed from1 hour to 8 weeks after step (a), or wherein step (a) is performed from1 hour to 8 weeks after step (b). In further embodiments, the methodfurther comprises (c) administering to the human subject an effectiveamount of a second antigenic peptide immunization composition, whereinthe second antigenic peptide immunization composition comprises the sameantigenic peptide immunization composition as used in (a). In furtherembodiments, the method further comprises administering an adjunctivetherapy selected from surgery, chemotherapy, radiation therapy, antibodytherapy, immunosuppressive therapy, or any combination thereof, such ascyclophosphamide, trastuzumab, anti-PD1, anti-PDL1, anti-CTLA4, or anycombination thereof. In further embodiments, the the elicited immuneresponse treats FRα-associated cancer.

Accordingly, cancers that may be treated using the compositions andmethods disclosed herein include breast cancer, triple negative breastcancer, inflammatory breast cancer, ovarian cancer, uterine cancer,colorectal cancer, colon cancer, primary peritoneal cancer, testicularcancer, renal cancer, melanoma, glioblastoma, lung cancer, or prostatecancer.

Effective countermeasures to biological pathogens are a criticalcomponent of biodefense and national security (Altmann, Expert RevVaccines 4, 275-279, 2005), and additional products are needed to meetbiodefense needs (Matheny, J., Mair, M. & Smith, B. Nat Biotechnol 26,981-983, 2008; Cohen, J. Science 333, 1216-1218, 2011; Artenstein, A. W.& Grabenstein, J. D. Expert Rev Vaccines 7, 1225-1237, 2008). Vaccinesprovide not only preventive or therapeutic countermeasures, but can alsoserve as actual deterrents (Poland et al., Vaccine 27, D23-D27, 2009) inthat they can be rapidly deployed to negate the primary outcomes ofbiological terrorism and thereby removing the motivation to use abioweapon.

For a vaccine composition incorporating nucleic acid molecules asdescribed herein, the desired end result is a safe product capable ofinducing long-lasting, protective immunity with minimal side effects,and as compared to other strategies (e.g., whole live or attenuatedpathogens), is inexpensive to produce, will minimize or eliminatecontraindications that have otherwise (typically) been associated withthe use of whole or attenuated virus vaccine compositions, and have anextended shelf-life because it is nucleic acid and/or syntheticpeptide-based. The ability to rapidly respond to infectious diseaseemergencies (natural outbreaks, pandemics, or bioterrorism) is a benefitof an effective use of the embodiments disclosed herein, whether incontext of biodefense or cancer immunotherapies or technologies. Theinstant disclosure sets forth the rapid identification and selection ofrelevant pathogen-related or cancer-related antigenic epitopes forpeptide-based vaccines and nucleic acid-based vaccines and is easilyadaptable to multiple Category A-C agents, as well as new and emergingpathogens.

In other embodiments, a chimeric nucleic acid molecule encodes anantigen or antigenic epitope from a pathogen. In certain embodiments,the pathogen is a virus, parasite, or bacteria. In further embodiments,a chimeric nucleic acid molecule encodes a PAA from one or morepathogens.

In certain embodiments, a PAA comprises an antigen from a virus, whereinthe virus is a small pox virus and other related pox viruses,lentiviruses such as HIV, influenza virus, Arenaviruses such as Junin,Machupo, Guanarito, Chapare, Lassa, and Lujo, Bunyaviruses such asHantaviruses, Rift Valley Fever virus, and Crimean Congo HemorrhagicFever virus, Flaviruses such as Dengue fever virus, Filoviruses such asEbola and Marburg virus, retrovirus, adenovirus, parvovirus,coronavirus, rhabdovirus, paramyxovirus, picornavirus, alphavirus,adenovirus, herpesvirus, Norwalk virus, togavirus, reovirus, papovarius,hepadnavirus, hepatitis virus, avian leucosis-sarcoma, mammalian C-typeviruses, B-type viruses, D-type viruses, HTLV-BLV group viruses, orspumavirus. In some embodiments, the influenza virus is influenza strainH7N9 (bird flu), H5N1, H3N8, H2N2, H3N2, H3N3, H9N2, H7N7, H1N1, or acombination thereof.

In other embodiments, a PAA comprises an antigen from a bacteria. Incertain embodiments, the bacteria is a Mycobacterium tuberculosis,pathogenic Escherichia coli, Yersinia pestis, Listeria monocytogenes,Clostridium botulinum, Bacillus anthracis, Staphylococcus aureus,Streptococcus pyogenes, Streptococcus pneumoniae, or Salmonellaenterica. The pathogenic E. coli can be E. coli strain 0157:H7, ETEC,EPEC, EIEC, EHEC, EAEC, or a combination thereof.

In certain embodiments, a PAA comprises an antigen from a parasite. Insome embodiments, the parasite is a Protozoa such as Plasmodium sp.,Entamoeba, Giardia Trypanosoma brucei, Toxoplasma gondii, Acanthamoeba,Leishmania, Babesia, Balamuthia mandrillaris, Cryptosporidium,Cyclospora, or Naegleria fowleri, a Parasitic worms such as Guinea worm(Dracunculus), Ascaris lumbricoides, Pinworm, Strongyloides stercoralis,Toxocara, Guinea Worm, Hookworm, Tapeworm, or Whipworm, or a Parasiticfluke such as Schistosoma, Gnathostoma, Paragonimus, Fasciola hepatica,or Trichobilharzia regent.

In any of the embodiments disclosed herein, a nucleic acid sequenceencoding the antigen can be codon optimized for expression in theappropriate subject (e.g., human). In certain embodiments, a smallpoxnucleic acid sequence comprises a smallpox MTI-PAA encoding an antigenset forth in SEQ ID NOS.:24-30, or any combination thereof. In someembodiments, a nucleic acid sequence encoding a smallpox MTI-PAAcorresponds to the sequence set forth in SEQ ID NO.:7. In furtherembodiments, a nucleic acid sequence encoding a smallpox MTI-PAAcorresponds to a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence setforth in SEQ ID NO.: 7.

In certain embodiments, a nucleic acid vector containing a multiplextranslation leader sequence portion (or PolyStart™) may also contain anucleic acid sequence encoding a TAP protein. An encoded TAP may be as aheterodimer (TAP1 plus TAP2) or only TAP1 or only TAP2. A TAP may becodon optimized so that it can be distinguished from an endogenous TAP.In addition, a TAP translation product may incorporate a peptide tagdetection moiety/portion such as a VSV G protein epitope tag, an HAepitope tag, a Myc epitope tag or any other detection portion.

Accordingly, in addition to the MTI-PAA, the chimeric nucleic acidmolecule disclosed herein can further include a nucleic acid moleculeencoding a TAP protein. In some embodiments the TAP protein is TAP1,TAP2, TAPL, or any combination thereof. In certain embodiments, the TAPprotein is a heterodimer of TAP1 and TAP2. In some embodiments, thenucleic acid molecule encoding the TAP protein has a polynucleotidesequence as set forth in any one of SEQ ID NOS.:206-210, or anycombination thereof.

In some embodiments, the chimeric nucleic acid disclosed herein isincluded in a nucleic acid vector. In some embodiments, the vector is anexpression vector. Examples of an expression vector include a plasmid, acosmid, a viral vector, an RNA vector, or a linear or circular DNA orRNA molecule. In some embodiments, the vector is a plasmid and comprisespcDNA3, pSG5, pJ603, or pCMV. In some embodiments, the vector is a viralvector selected from a retrovirus, adenovirus, parvovirus, coronavirus,influenza virus, rhabdovirus, paramyxovirus, picornavirus, alphavirus,adenovirus, herpesvirus, poxvirus, Norwalk virus, togavirus, flavivirus,reovirus, papovarius, hepadnavirus, hepatitis virus, avianleucosis-sarcoma, mammalian C-type viruses, B-type viruses, D-typeviruses, HTLV-BLV group viruses, lentivirus, or spumavirus. In the caseof a viral vector incorporating a nucleic acid molecule of thisdisclosure, a virus may be a DNA virus (e.g., vaccinia, adenovirus) oran RNA virus (e.g., vesicular stomatitis virus). In certain embodiments,a virus is oncolytic.

In certain embodiments, a multiplex vector of the instant disclosure hasa nucleic acid molecule that encodes an MTI-PAA fusion operably linkedto a CMV promoter and the fusion protein contains a carboxy-terminalVSV-g epitope tag (SEQ ID NO.:13). In further embodiments, a multiplexvector of the instant disclosure includes a Tap1 gene expressed by anSV40 promoter and the TAP1 includes an amino-terminal V5 epitope tag(SEQ ID NO.:14), while a nucleic acid encoding an MTI-PAA is operablylinked to a CMV promoter and a carboxy-terminal VSV-g epitope tag (SEQID NO.:13). In still further embodiments, a multiplex vector of theinstant disclosure includes a Tap2 gene expressed by an SV40 promoterand the TAP2 includes an amino-terminal AU5 epitope tag (SEQ ID NO.:15),while a nucleic acid molecule encoding an MTI-PAA is operably linked toa CMV promoter and the PAA included a carboxy-terminal VSV-g epitope tag(SEQ ID NO.:13). In yet further embodiments, a multiplex vector of theinstant disclosure includes a Tap1 and Tap2 gene separated by an IRESand operably linked to an SV40 promoter, wherein the TAP1 includes anamino-terminal V5 epitope tag (SEQ ID NO.:14) and the TAP2 includes anamino-terminal AU5 epitope tag (SEQ ID NO.:15), while a nucleic acidmolecule encoding an MTI-PAA is operably linked to a CMV promoter andthe PAA included a carboxy-terminal VSV-g epitope tag (SEQ ID NO.:13).

A class I peptide vaccines incorporated into a nucleic acid-basedimmunization composition can elicit protective immunity, and theinclusion of HLA class II peptides and the generation of T helperresponses is expected to stimulate optimal cellular immunity and augmentthe protection provided by CTL alone. In some embodiments, a nucleicacid of the instant disclosure encoding one or more Class II ‘reporterepitope’ PAA and class II epitope-containing PAAs may be represented asa MTI-VSVG-PAA, wherein the MTI portion is mutated to remain cytosolic.As designed, a MTI-VSVG-PAA results in a primary translation productcontaining what is referred to herein as an internalized VSVG signalsequence (e.g., SEQ ID NO.: 42). The internalized signal sequence isprocessed and the chimeric protein secreted or membrane localized,thereby facilitating entry of the PAA into the endocytic compartment andclass II processing pathway. VSVG processing and signaling sequences arediscussed in greater detail in Rose et al, J. Virol. 39: 519-528, 1981;Gallione et al. J. Virol. 54:374-382, 1985; Machamer et al., Mol. Cell.Biol. 5:3074-3083, 1985; Rottier et al. J. Biol. Chem. 262:8889-8895,1987, all of which are herein incorporated by reference in theirentirety.

Accordingly, in some embodiments is a chimeric nucleic acid disclosed inany of the embodiments herein further comprises an internalization VSVGsignal sequence, a VSVG secretion signal sequence, a traffickingsequence, a dendritic cell targeting sequence, a membrane localizationsequence, or any combination thereof. In some embodiments, the chimericnucleic acid comprises an internalization VSVG signal sequence as setfor in SEQ ID NO.: 43. In some embodiments, the nucleic acid comprises aVSVG secretion signal sequence as set for SEQ ID NOS.:47 or 48. The VSVGsecretion signal sequence promotes trafficking through ER/Golgi to thecell surface (i.e., plasma membrane). In some embodiments, the nucleicacid comprises a dendritic cell targeting sequence as set for in SEQ IDNO 45. The DC targeting sequence is optional, and may be placed at theCOOH terminus. In some embodiments, the nucleic acid comprises a VSVGmembrane localization sequence as set for in SEQ ID NO.:57. In someembodiments, the nucleic acid comprises a PAA including sequencesencoding folate receptor-α Class II antigens and is a secreted chimera.In some embodiments, the nucleic acid comprises a sequence as set forthin SEQ ID NO.:52. In some embodiments, the nucleic acid comprises a PAAincluding sequences encoding folate receptor-α Class II antigens and isa membrane localized polypeptide. In some embodiments, nucleic acidcomprises a PAA including sequences encoding folate receptor-α Class IIantigens and is a membrane localized polypeptide such as the fusionprotein encoded by the nucleic acid sequence as set forth in SEQ IDNO.:58.

The chimeric nucleic acid and vectors described herein can be introducedinto a cell. Methods of introducing nucleic acid molecules are wellknown in the art and include transformation and transduction.Accordingly, some embodiments comprise a cell that includes a nucleicacid molecule as described herein. In addition, certain embodimentscomprise a cell that includes a vector according to any of theembodiments described herein. In some embodiments, the cell is anautologous cell obtained from a first subject or an allogeneic cell froma subject different from the first subject. The cell can be located invitro or in vivo. In some embodiments, the cell is an antigen presentingcell, such as a professional or non-professional antigen presentingcell. The antigen presenting cell can be a professional antigenpresenting cell such as a dendritic cell or a macrophage. In a preferredembodiment, the antigen presenting cell is a dendritic cell.

The term dendritic cells (DCs) was described by Steinman et al in 1973(Steinman R M and Cohn Z A J Exp Med 137:1142-62, 1973). Dendritic cellsare derived from myeloid bone marrow progenitor cells and have thepotential to be used as a viable cell-based anti-cancer therapy(Vacchelli et al., OncoImmunology 2:10, 2013; Slingluff et al., ClinCancer Res 12(7 Suppl):2342s-2345s, 2006; Steinman, Immunity 29:319-324, 2008). DCs localize to lymphoid tissues, skin (e.g., epidermalLangerhans cells) and various mucosae. When mature, DCs are potentprofessional antigen presenting cells for both HLA Class II as well asHLA Class I restricted systems (Santambrogio et al., PNAS96(26):15050-55, 1999). Mature DCs express elevated levels of HLA ClassII proteins on the cell surface, migrate to lymph nodes and secrete highlevels of cytokines/chemokines. T-cell activating antigenic peptidesbound to MHC Class II proteins presented on the cell surface of anactivated DC stimulate (activate) both cognate CD4⁺ helper T-cells andCD8⁺ cytotoxic T-cells, while at the same time secreting a number ofcytokines and other growth promoting factors. Subdermal administration(a site rich in DCs) of a peptide or nucleic acid-based composition setforth herein results the uptake and immune system presentation ofantigenic T-cell peptides (including naturally processed antigenicpeptides) in context with DC expressed HLA Class I or Class II proteins.

The affinity of a TAA-derived peptide or nucleic acid-based antigenicT-cell peptide composition, as disclosed herein, for a DC may beenhanced by inclusion of one or more DC targeting motif such as apolypeptide, small molecule, or antibody-based technology such as taughtin Diebold et al., Gene Therapy 8:487-493, 2001; Bonifaz et al., J ExpMed 196(12):1627-1638, 2002; Birkholz et al., Blood 116(13):2277-2284,2010; Apostolopoulos et al., J. Drug Delivery, p 1-22, 2013; Lewis etal., Biomaterials 33(29):7221-7232, 2012; Gieseler et al., ScandinavianJ Immunol. 59: 415-424, 2004, all of which are herein incorporated byreference in their entirety. For example, DC affinity may be enhanced byincluding one or more antibodies or other molecules with affinity to DCsurface markers such as DEC-205, DC-SIGN, CLEC4A, and may also include amaturation signal, e.g., IL-15.

In contrast to conventional polypeptide based vaccines, DNA vaccines maycomprise a nucleic acid in the form of a plasmid (Li et al., J.Biotechnol. 162:171, 2012) but may also be incorporated in the form ofRNA or incorporated into the nucleic acid of a virus vector fordelivery. The plasmid DNA includes a promoter driving expression of oneor more transcription units set forth herein, as would be appreciated byone of ordinary skill in the art. A nucleic acid based vaccine can beadministered by, for example, intramuscular injection, subcutaneously,intranasally, via mucosal presentation, intravenously or by intradermalor subcutaneous administration.

A nucleic acid based vaccine is administered to a patient (either usinga DNA plasmid, a RNA, or a viral vector) whereby the nucleic acid istaken up into a cell's cytoplasm and/or nucleus where it is transcribedinto mRNA and then translated into a polypeptide or protein. Vaccinemodalities set forth herein may be administered individually orsequentially as a prime-and-boost platform. The priming vaccinecomposition may be peptide-based followed by a vaccine boost comprisinga nucleic acid delivered, for example, as a plasmid DNA or as a viraldelivery system. Alternately, the priming vaccine composition may benucleic acid delivered followed by a peptide-based vaccine. Either thevaccine prime or boost, or both the prime and boost, may be administeredonce or multiple times to a patient in need thereof.

Accordingly, in some embodiments are methods of eliciting a cellularimmune response, comprising administering to a subject an effectiveamount of an immunization composition comprising a nucleic acidmolecules including an MTI-PAA as described in any of the embodimentsherein, an antigen encoded by a nucleic acid molecule according to anyof the embodiments herein, or both, thereby eliciting a cellular immuneresponse. In some embodiments, the immunization composition comprises anucleic acid immunization composition. In some embodiments, theimmunization composition comprises an antigen or polyantigenimmunization composition. In some embodiments, the nucleic acidimmunization composition and the antigen immunization composition areboth administered. In certain embodiments, the antigen immunizationcomposition is administered first and the nucleic acid immunizationcomposition is administered second. In other embodiments, the nucleicacid immunization composition is administered first and the antigenimmunization composition is administered second. In yet furtherembodiments, the nucleic acid immunization composition and the antigenimmunization composition are administered concurrently. In someembodiments, the immunization composition comprises a cell comprising anucleic acid according to the embodiments disclosed herein. In someembodiments, the cellular immune response is an antigen-specific T-cellresponse. In some embodiments, the subject is human. In certainembodiments, the nucleic acid immunization composition is taken up by anantigen presenting cell. The antigen presenting cell can be aprofessional or non-professional antigen presenting cell. In someembodiments, the antigen presenting cell is a dendritic cell. In certainembodiments, the method comprises administering a nucleic acidimmunization composition to a subject wherein the nucleic acidimmunization composition comprises a chimeric nucleic acid moleculeincluding an MTI as described herein (e.g., any of SEQ ID NOS.:1-6, 95,or 96) and including a PAA wherein the PAA comprises antigens fromfolate receptor-α (e.g., any of SEQ ID NOS.:69-93), Her2/neu (e.g., anyof SEQ ID NOS.:117-135), or any combination thereof. Exemplary folatereceptor-α PAA and MTI-PAA sequences are provided in SEQ ID NOS.:49, 50,52, 53-55, 58-64, 66-68, and 97-100. Exemplary HER2/neu PAA and MTI-PAAsequences are provided in 115, 116, 136-150.

The MTI technology described herein may also be used to modify apatient's cells ex vivo. For example, isolated or enriched preparationsof a patient's T-cells, dendritic cells, or other antigen presentingcell population may be transfected with a nucleic acid molecule asdisclosed herein. Transfected cells may be expanded and thenreintroduced into the patient as effector T-cells.

Therefore, in certain embodiments, the method of eliciting a cellularimmune response includes contacting a cell with a nucleic acidimmunization composition comprising the chimeric nucleic acid moleculeas described in any of the embodiments herein, wherein the cell iscontacted ex vivo and then administered to a subject. Methods ofisolating cells from a subject and introducing nucleic acid moleculesare known in the art. In some embodiments, the cell is autologous orallogeneic. In certain embodiments, the cell is an antigen presentingcell. In some embodiments, the cell is a dendritic cell. In someembodiments, the subject is a human.

In certain embodiments, the instant disclosure provides a method ofeliciting a cellular immune response, comprising administering to asubject an effective amount of a cell comprising a nucleic acid moleculeor a vector of any one of the embodiments disclosed herein, therebyeliciting a cellular immune response. In some embodiments, a cell isautologous or allogeneic. In some embodiments, a cell is an antigenpresenting cell. In certain embodiments, the antigen presenting cell isa dendritic cell. In some embodiments, the subject is a human.

In some embodiments, the present disclosure provides a method for primeand boost to elicit a robust immune response that results in theproduction of memory T cells. For example, an immune response iselicited against a cancer or infectious disease by (a) contacting thesubject with a nucleic acid molecule immunization composition asdescribed herein, wherein the nucleic acid molecule of the nucleic acidmolecule immunization composition encodes one or more antigenicpeptides, (b) optionally allowing a time sufficient to generate aninitial immune response, (c) contacting a subject with an antigenicpeptide immunization composition. The peptide immunization compositionand the nucleic acid immunization composition can correspond to the sameone or more HLA Class I peptide antigen(s). Alternatively, the peptideimmunization composition and the nucleic acid immunization compositioncan correspond to the same cancer marker protein or TAA.

In certain embodiments, provided herein is a method of eliciting acellular immune response, comprising (a) administering to a subject aneffective amount of an antigen immunization composition comprising oneor more antigens encoded by any one of the nucleic acid moleculesdisclosed herein, and (b) administering to the subject an effectiveamount of a nucleic acid molecule immunization composition comprising anucleic acid molecule or vector of any one of the embodiments describedherein. In some embodiments, the nucleic acid molecule immunizationcomposition of step (b) encodes a plurality of antigens. In someembodiments, the antigen immunization composition of step (a) comprisesa plurality of antigens. In certain embodiments, the nucleic acidmolecule encodes one or more antigens from the same protein as the oneor more antigens used in step (a). In some embodiments, the nucleic acidmolecule of the nucleic acid molecule immunization composition encodesone or more of the same antigens as the one or more antigens used instep (a). In certain embodiments, step (b) is performed simultaneouslywith step (a). In other embodiments, step (b) is performed sequentiallyto step (a). In some embodiments, step (b) is performed from 1 hour to 5months, from 1 hour to 4 months, from 1 hour to 3 months, from 1 hour to8 weeks, from 1 hour to 6 weeks, from 1 hour to 4 weeks, from 1 hour to3 weeks, from 1 hour to 2 weeks, from 1 hour to 1 week, from 1 hour to72 hours, from 1 hour to 48 hours, or from 1 hour to 24 hours after step(a). In other embodiments, step (a) is performed 1 hour to 5 months,from 1 hour to 4 months, from 1 hour to 3 months, from 1 hour to 8weeks, from 1 hour to 6 weeks, from 1 hour to 4 weeks, from 1 hour to 3weeks, from 1 hour to 2 weeks, from 1 hour to 1 week, from 1 hour to 72hours, from 1 hour to 48 hours, or from 1 hour to 24 hours after step(b). In some embodiments, the method further comprising (c)administering to the subject a second effective amount of an antigenimmunization composition, wherein the antigen immunization compositioncomprises a peptide that is derived from the same protein as theantigenic peptide used in (a). For example, in some embodiments theantigen immunization composition of step (c) is the same as the antigenimmunization composition used in (a).

In other embodiments, provided herein is a method of eliciting ancellular immune response, comprising (a) administering to a subject aneffective amount of a nucleic acid molecule immunization compositioncomprising the nucleic acid molecule of any of the embodiments describedherein, (b) allowing a time sufficient to generate an initial immuneresponse, and (c) administering to the subject a second effective amountof a nucleic acid molecule immunization composition comprising a nucleicacid molecule according to any of the embodiments described herein, oran effective amount of an antigenic peptide immunization composition. Insome embodiments, the antigen immunization composition comprises one ormore antigens that are from the same protein as the antigen encoded bythe nucleic acid molecule of step (a). In some embodiments, the nucleicacid molecule of the nucleic acid molecule immunization compositionencodes one or more of the same antigenic peptides as used in (c). Incertain embodiments, the time sufficient to generate an initial immuneresponse is from 1 hour to 5 months, from 1 hour to 4 months, from 1hour to 3 months, from 1 hour to 8 weeks, from 1 hour to 6 weeks, from 1hour to 4 weeks, from 1 hour to 3 weeks, from 1 hour to 2 weeks, from 1hour to 1 week, from 1 hour to 72 hours, from 1 hour to 48 hours, orfrom 1 hour to 24 hours after step (a). In certain embodiments, themethod further comprising (d) administering to a subject an effectiveamount of an antigen immunization composition, wherein the antigenimmunization composition comprises one or more antigens that are fromthe same protein as the antigen used in (c). In some embodiments, theantigen immunization composition of step (d) comprises the same antigenused in (c).

In certain embodiments, a nucleic acid molecule immunization compositionand a antigen immunization composition encode a different peptideantigen class, for example peptide(s) of a peptide immunization complexmay comprise one or more HLA Class II antigenic peptide epitopes derivedfrom a cancer marker protein, while the nucleic acid molecule may encodeone or more HLA Class I antigenic peptides derived from the same cancermarker protein (e.g., Her2/neu or folate receptor alpha).

Antigenic peptide epitopes contained within a nucleic acid immunizationcomposition and antigenic peptide epitopes contained within a peptideimmunization composition may be derived from more than one cancer markerprotein (e.g., Her2/neu and folate receptor alpha).

Typically, a patient having cancer is incapable of eliciting a strongenough immune response that can destroy sufficient numbers of cancercells to either eliminate the tumor entirely or sufficient to reducetumor burden to a manageable level and thereby provide an improvedstandard of living, with a long time to recurrence. The health careoptions to patients with cancer have historically focused on surgicalresection of the tumor mass, chemotherapy or radiation therapy. In eachof these historical options, the treatment is particularly invasive orparticularly indiscreet in that chemotherapies also kill a patient'sgood cell as well as unwanted cancer cells.

An advantage of stimulating a patient's own immune system to destroycancer cells is that, in general accordance with the embodiments setforth herein, such immune stimulation is long lived and consequentlyshould prevent or extend the time to recurrence. In contrast, radiationtherapy or chemotherapy requires continued repetitive treatment in orderto keep killing unwanted cancer cells. Stimulating a patient's ownimmune system cells may be mediated in vivo or ex vivo. For an ex vivoadministration, a patient's cells (e.g., dendritic cells and/or T-cells)are removed from the patient then contacted with a peptide, nucleic acidor viral composition of the instant invention. After contacting, thepatient's cells are administered back to the patient.

A vaccine approach as described herein is designed to stimulate apatient's immune cells to function for a long time and therefore if anew cancer cell comes into existence it will be destroyed. In oneembodiment, long term immune function is accomplished using amulti-antigenic peptide composition capable of stimulating bothcytotoxic CD8⁺ T-cells (HLA Class I restricted) and helper CD4⁺ T-cells(HLA Class II restricted). Thereby, the patient does not need torepeatedly go to their health care provider (doctor, hospital) foranother round of chemotherapy or radiation therapy. In many situationsin which the treatment with chemotherapy, radiation therapy or surgery,the treatment can be an obstacle to success, indeed, often the patientfeels worse as a consequence of treatment rather than the disease(cancer) itself.

In some embodiments, the methods of eliciting a cellular immune responsedescribed above are to elicit an immune response against a TAA asdescribed herein. In other embodiments, the methods of eliciting acellular immune response described above are to elicit an immuneresponse against a pathogen as described herein. In any of the methodsof eliciting a cellular immune response described above, the subject canbe a human.

In any of the methods of eliciting a cellular immune response describedabove, the method can further comprise administering an adjunctivetherapy. In some embodiments, the adjunctive therapy is surgery,chemotherapy, radiation therapy, antibody therapy, or a combinationthereof. In some embodiments, the adjunctive therapy iscyclophosphamide.

Cancer, for example breast cancer, is diagnosed in approximately 210,000women each year. Conventional standards of care such as surgery,chemotherapy and radiation therapy are successful treatments at leastinitially however recurrence is a common problem and is frequently themain source of morbidity and mortality. Monoclonal antibodies have beenadvanced in the treatment of some cancers, for example trastuzumab forHER2/neu+ breast cancer. Although the combination of trastuzumab extendssurvival time for women with advanced HER2/neu+ cancer, a majority ofwomen develop resistance within one year of the beginning of treatment.The development of additional or alternative strategies may providepatients with new treatment options and improve the current standard ofcare. A vaccine that delays the time to disease recurrence or preventsdisease recurrence has significant clinical and commercial potential. Inaddition, a cancer vaccine described herein may be used to boostimmunity against tumor antigenic T-cell epitopes that are known orexpected to generate pre-existent immunity towards a TAA detected in acancer patient.

In other embodiments, provided herein is a method of treating breastcancer, comprising administering an effective amount of nucleic acidmolecule immunization composition comprising a nucleic acid moleculeimmunization composition comprising a breast cancer TAA. In someembodiments, the breast cancer TAA is HER2/neu, folate receptor-α, or acombination thereof. In some embodiments, the nucleic acid immunizationcomposition comprises a nucleic acid sequence corresponding SEQ IDNOS.:49, 50, 52-55, 58-64, 66-93, 97-100, 115-150, or any combinationthereof. In certain embodiments, the method of treating breast cancerfurther comprises administering an adjunctive therapy. The adjunctivetherapy can be surgery, chemotherapy, radiation therapy, antibodytherapy, or any combination thereof. In some embodiments, an antibodytherapy comprises trastuzumab, pertuzumab, anti-CTLA4, anti-PD1,anti-PDL1, anti-VEGF (e.g., bevacizumab), anti-Folate Receptor alpha(e.g., farletuzumab); as well as small molecule inhibitors of kinasedomain function (e.g., laptinib, gefitinib, erlotinib, or the like). Insome embodiments, the adjunctive therapy is cyclophosphamide. In someembodiment, the adjunctive therapy is therapy that inhibitsimmunosuppression. In certain embodiments, the therapy that inhibitsimmunosuppression, is an inhibitor of an immunosuppression signal is anantibody, fusion protein, or siRNA specific for PD-1, PD-L1, PD-L2,CTLA4, HVEM, BTLA, KIR, LAG3, GALS, TIM3, TGFβ, IL-10, IL-35, or anycombination thereof. In some embodiments, the anti-CTL4 antibody is anCTLA4 specific antibody or binding fragment thereof, such as ipilimumab,tremelimumab, CTLA4-Ig fusion proteins (e.g., abatacept, belatacept), orany combination thereof. In some embodiments, the anti-PD-1 antibody isa PD-1 specific antibody or binding fragment thereof, such aspidilizumab, nivolumab, pembrolizumab, MK-3475, AMP-224, or anycombination thereof. In some embodiments, the anti-PD-L1 antibody is aPD-L1 specific antibody or binding fragment thereof, such as MDX-1105,BMS-936559, MEDI4736, MPDL3280A, MSB0010718C, or any combinationthereof.

In some embodiments, the methods of eliciting a cellular immune responseas described herein further comprise identifying patient specific TAAantigens. Accordingly a population of a patient's T-cells can bescreened against a collection of Class I and/or Class II T-cell peptidesin order to identify antigenic epitopes to which the patient already hasa detectable T-cell immune response. A patient's cells are isolated,incubated with peptides displayed using a multi-well plate and cytokineresponses are measured, such as gamma interferon. One or more of theantigenic peptides so identified can then be formulated into apeptide-based vaccine composition. The detected antigenic peptides maybe Class I or Class II restricted. In addition, the same Class I and/orClass II peptides may be incorporated into a nucleic acid-based deliverysystem disclosed herein. A patient may then be administered the tailoredpeptide or corresponding nucleic acid based compositions as standalonemedicines or in sequential combination as a prime-and-boost modality asdescribed in embodiments above. In this regard, the prime may be nucleicacid based and the boost peptides-based, or vice versa. Accordingly, insome embodiments is a method of treating cancer, comprisingadministering to a subject an effective amount of a nucleic acidimmunization composition comprising a nucleic acid molecule of any ofthe embodiments described herein, wherein the one or more antigensencoded by the nucleic acid molecule comprise an antigen having anoncogenic mutation identified in the subject.

Supporting of the instant disclosure, compositions and methods describedherein combine mass spectrometry to aid in the identification of novelpeptide epitopes presented by HLA class I and HLA class II alleles.After administration, one of ordinary skill in the art can follow andcharacterize immune responses using cell culture and small animal ornon-human primate model systems. Peptide sequences are converted into anucleic acid sequence (which may be fully or partially codon optimized)and designed for inclusion into nucleic acid-based expression/deliveryvaccine systems. Exemplary peptides may be from cancer (e.g., HER2neupeptides such as those disclosed in US Patent Pub. No. 2010/0310640,which peptides are incorporated herein by reference; folate receptor-αpeptides; NY-ES01 testes specific antigens; WT1 peptides, or the like)or other infectious diseases (e.g., other viruses, parasites, bacteria).

The compositions and methods of the instant disclosure may include amolecular adjuvant mechanism (TAP1/TAP2) that is suited to enhance HLAclass I peptide presentation and subsequent CD8 T cell responses andcombines it with a vaccine vector designed to maximize antigenexpression (FIG. 1) incorporating features that enhance translationefficiency; induces production of an array of antigenic peptides; andmarks peptides for proteasome targeting/trafficking and proteolyticprocessing. The compositions and methods of the instant disclosure mayincorporate multiple translation initiation sites for increasedexpression (for example, one mRNA initiates and synthesizes fourtranslation products); incorporate cytosolic and/or nuclear targetingand subsequent processing; distinguish between endogenous andrecombinant protein; distinguish between endogenous and recombinantnucleic acid encoding a TAP1 and/or a TAP2; and allow for nuclear andcytosolic antigen targeting. The vectors may also be constructed suchthat each feature can be independently manipulated for characterizationand testing.

Additional immune stimulatory or modulating agents, including negativeimmune modulators such as regulatory T-cells and related check pointinhibitors (anti-PD1, Yervoy®, cyclophosphamide), may be included in anycomposition or method described herein. For example,granulocyte-macrophage colony-stimulating factor (GM-CSF) may functionas a vaccine adjuvant as previously described (see Mohebtash et al.,Clin. Cancer Res. 17:7164, 2011). Other adjuvants such as alum, MF59,CpG, R848 and the like may be included in the compositions or methodsdescribed herein.

As an example, one infectious disease treatable by administering theinstant invention is smallpox as this disease has claimed hundreds ofmillions of lives in the last two centuries alone (Dixon, C. W.Smallpox. (J. & A. Churchill, 1962); Fenner et al., Smallpox and itsEradication, Vol. 6, World Health Organization, 1988), and althoughconsidered eradicated since 1980 (Fenner, 1988), biodefense researchinto poxviruses remains vitally important because of the following: (1)concerns about the use of smallpox as a biological weapon (Henderson etal., Working Group on Civilian Biodefense. JAMA 281:2127-2137, 1999;Kennedy et al., Vaccine 27(Suppl 4):D73-79, 2009; Whitley, Antiviral Res57:7-12, 2003; Bossi et al., Cell Mol Life Sci 63:2196-2212, 2006; Mayr,Comp. Immunol. Microbiol. Infect. Dis. 26:423-430, 2003; Wiser et al.,Vaccine 25:976-984, 2007); (2) emerging zoonotic poxviruses, such asmonkeypox in the US and Africa (Hutson et al., Am. J. Trop. Med. Hyg.76:757-768, 2007; Kile et al., Arch. Ped. Adolesc. Med. 159:1022-1025,2005; Di Giulio and Eckburg, The Lancet Infect. Dis. 4:15-25, 2004;Edghill-Smith et al., J Infect. Dis. 191:372-381, 2005; Larkin, TheLancet Infect. Dis. 3:461, 2003; Jezek et al., Am. J. Epidemiol.123:1004, 1986), vaccinia-like viruses in South America (Leite et al.,Emerging Infect. Dis. 11:1935-1938, 2005; Silva-Fernandes et al., Clin.Virol. 44:308-313, 2009; Trindade et al., Clin. Infect. Dis. 48:e37-40,2009), and buffalopox in India (Singh et al., Animal Health Res.Rev./Conference of Research Workers in Animal Diseases 8:105-114, 2007);(3) numerous vaccine contraindications (Neff et al., Clin. Infect. Dis.46(Suppl 3):5258-270, 2008; Poland et al., Vaccine 23:2078-2081, 2005;Bonilla-Guerrero & Poland, J Lab. Clin. Med. 142:252-257, 2003); (4)concerns with VACV transmission (Fulginiti et al., Clin. Infect. Dis.37:241-250, 2003; Wharton et al., MMWR Recomm. Rep. 52:1-16, 2003;Redfield et al., N. Engl. J Med. 316:673-676, 1987); and (5) safetyissues inherent to live virus smallpox vaccines resulting in rare butserious adverse events (Fulginiti et al., Clin. Infect. Dis. 37:251-271,2003; Fulginiti, JAMA 290:1452; author reply 1452, 2003; Lane et al.,JAMA 212:441-444, 1970; Lane et al., N. Engl. J. Med. 281:1201-1208,1969; Morgan et al., Clin. Infect. Dis. 46(Suppl 3):5242-250, 2008;Vellozzi et al., Clin. Infect. Dis. 39:1660-1666, 2004; Halsell et al.,JAMA 289:3283-3289, 2003; Poland and Neff, Immunol. Allergy Clin. NorthAm. 23:731-743, 2003; Chapman et al., Clin. Infect. Dis. 46(Suppl3):S271-293, 2008). Recently introduced second-generation (live) tissueculture-based vaccines (ACAM2000) have replaced Dryvax®, but retainnearly all of the drawbacks of the first-generation vaccines (Kennedy etal., Curr. Opin. Immunol. 21:314-320, 2009; Artenstein et al., Vaccine23:3301-3309, 2005; Greenberg and Kennedy, Expert Opin. Investig. Drugs17:555-564, 2008; Poland, Lancet 365:362-363, 2005; Greenberg et al.,Lancet 365:398-409, 2005). Attenuated vaccines based on MVA (IMVAMUNE)may have improved safety profiles (Kennedy and Greenberg, Expert RevVaccines 8:13-24, 2009; Frey et al., Vaccine 25:8562-8573, 2007; Vollmaret al., Vaccine 24:2065-2070, 2006; Edghill-Smith et al., J Infect. Dis.188:1181, 2003), but remain live viral vaccines and there are someconcerns regarding both safety and immunogenicity.

The preparation and characterization of expression vectors (plasmid andvaccinia virus based) encoding peptide antigen arrays (PAAs) consistingof multiple HLA Class I and Class II-derived peptides together with TAP1and/or TAP2 is set forth herein. As one example of the instantdisclosure, a plasmid expression vector encoding various combinations ofthe features outlined in FIG. 1: (1) a vaccinia-peptide antigen arrayand (2) an expression cassette containing TAP1 and/or TAP2. Theseexpression vectors direct the synthesis of a chimeric protein containingan amino-terminal portion of FGF2 (MTI) followed by an interchangeablepeptide antigen array containing a selection of peptides associated withinfectious disease or cancer.

In order to promote efficient proteasome processing of the encodedpeptide sequences and thereby preserve complete protease processingsites, the NH₂ and COOH ends can be extended by three or four or morenative/naturally occurring amino acids (FIG. 1, lower case). Eachantigenic peptide portion may be further separated from the nextantigenic peptide by a spacer (e.g., G₄S). The four MTI translationinitiation sites will yield four moles of a multi-peptide PAAtranslation product for every one mole of transcribed mRNA (FIG. 1)(Florkiewicz and Sommer, PNAS 86:3978-3981, 1989; Florkiewicz et al.,Growth Factors 4:265-275, 1991).

In accordance with the instant disclosure, nucleic acid molecule-basedexpression vectors have been designed so that a PAA portion of a MTI-PAAis a cassette that is easily excised and replaced. This enables one ofordinary skill to rapidly incorporate additional VACV/VARV epitopes,create a ‘reporter epitope’ PAA, or design PAAs specific to otherpathogens or cancer targets, consistent with a modular recombinantstrategy.

The COOH-terminal VSVG epitope-tag can be used for detection byimmunoprecipitation, immunoblotting and indirect immunofluorescence (andcan also be used to design PCR primer pairs for detection of transcribedmRNA). In order to enhance expression of antigenic peptides/proteinsalong with TAP1 and/or TAP2, nucleic acid molecule encoding TAP1, TAP2,and PAA (not MTI) are prepared synthetically and in context with apreferred codon utilization algorithm, thereby providing enhancedtranslation efficiency consistent with the preferred codon bias of Homosapiens, while at the same time minimizing potential cis-acting mRNAsequences that could negatively impact translation efficiency.

Furthermore, nucleic acids of this disclosure may incorporate commonepitope tags for the differential immune detection of vector-encoded, asopposed to endogenous Tap1 and/or Tap2, protein. The NH₂-terminal Tap1tag is V5 and the NH₂-terminal tag for Tap2 is AU5.

It is appreciated that proteasomes differ in composition and processingability (Bedford et al., Trends Cell Biol. 20:391, 2010; de Graaf etal., Eur J Immunol 41:926-935, 2011; Khan et al., J Immunol 167:6859,2001; Sijts & Kloetzel, Cellular and Molecular Life Sciences: CMLS 68,1491-1502, 2011; Wilk et al., Arch Biochem Biophys 383, 265, 2000; Xie,Y. J Mol Cell Biol 2, 308-317, 2010). Furthermore, it is recognized,although not well characterized, that nuclear proteasomes appeardifferent from cytosolic proteasomes. In accordance with the invention,MTI translation products initiated with CUG codons are targeted to thenucleus while the translation product initiated at the AUG codon remaincytosolic. By adjusting the MTI sequence (site specific or deletionmutagenesis remove multiple GR repeats), the invention disclosed canexclusively express either cytosolic or nuclear localized MTI-PAA.

The invention disclosed herein may include a molecular adjuvant, such asthe proteins termed Tap1 and/or Tap2, and can be used to demonstratethat inclusion of TAP1 can further enhance presentation of a PAA derivedpeptide.

In some embodiments, a ‘reporter epitope’ PAA consisting of epitopes forwhich there are peptide/MHC complex-specific antibodies may beincorporated into a PAA. Such a reporter epitope may allow one ofordinary skill to use these antibodies to directly measure surfaceexpression of the presented peptides in the context of their restrictingMHC/HLA allele.

Immunogenicity testing includes a series of dose-ranging andvaccination-schedule-testing experiments designed to characterizevaccine-induced immune responses. Different HLA class I and HLA class IItransgenic animal strains with matching HLA restriction to the antigenicepitopes may be used within the context of the disclosed invention.

Vaccine efficacy testing includes evaluation of the PAA/TAP vectorvaccination dose/schedule followed by survival studies using, forexample, the intranasal infection model. Intranasal inoculation ofVaccinia Western Reserve (WR) into mice results in a lethal infectioncharacterized by weight loss, ruffled fur, lethargy, and death by daysix or seven post infection (Turner, J Gen Virol 1:399, 1967; Reading &Smith J. Gen. Virol. 84:1973, 2003; Williamson et al., J. Gen. Virol.71:2761, 1990).

Prior to the vaccine efficacy experiments, the VACV-WR LD₅₀ for eachtransgenic strain may be determined experimentally as per theReed-Muench method, and 5-10 LD₅₀ will be used for survival experiments,and to develop correlates of protection/immunity to derive levels ofprotection in a human vaccinated population (Reed & Muench, Am. J.Epidemiol. 27:493, 1938).

CTL function: as appreciated by one of ordinary skill in the relevantart, percent specific lysis is calculated for each effector:target ratioaccording to established protocols (Latchman, Y. E. et al. PNAS 101,10691-10696, 2004) and compared between groups using ANOVA.

T helper function: Antigen-specific T cell activity is calculated bysubtracting the median IFNγ ELISPOT response value of the unstimulatedwells from that of the stimulated wells. Values may be calculated forindividual mice and differences between immunized and control groupswill be compared using Student's t test. CFSE a tracking reagent used inflow cytometry is used to determine proliferation rate differencesbetween groups and compared using established models (Banks et al., J.Immunol. Methods 373:143-160, 2011; Banks et al. Bull. Math Biol.73:116-150, 2011).

Survival studies: Power calculations (assuming a type I error rate of0.05 and a two-sided test of hypothesis) indicate that, with 20mice/group, about an 80% power of detection for a difference in survivalwill be measurable if the true survival rates at seven days are 50% forthe immunized group and 10% for the unimmunized group. For all survivalexperiments, Kaplan-Meier survival curves will be plotted and comparedusing standard statistical tests for survival, such as the log ranktest.

Proteasome processing of a PAA modified to include a consensus proteinubiquitination motif (e.g., KEEE (SEQ ID NO.: 203) or EKE) (Catic etal., Bioinformatics 20:3302-3307, 2004; Chen, Z. et al. PLoS One 6,e22930, 2011; Jadhav, T. & Wooten, M. W. J Proteomics Bioinform 2, 316,2009; Sadowski, M. & Sarcevic, B. Cell Div 5, 19, 2010) may be includedin the embodiments listed herein along with fluorescently-labeledantibodies specific for peptide/MHC complexes in order to directlymeasure epitope presentation.

In certain embodiments, TAP is selected as a molecular adjuvant toenhance class I antigen presentation. By way of background, there isconsiderable cross-talk between the HLA class I and class II processingand presentation pathways (Chicz, R. M. et al. The Journal ofexperimental medicine 178, 27-47, 1993; Lechler, R., Aichinger, G. &Lightstone, L. Immunological reviews 151, 51-79, 1996; Rudensky, A., etal., Nature 353, 622-627, 1991). In addition, intracellular peptides canbe loaded onto HLA class II peptides through multiple mechanisms(Dengjel et al., PNAS 102:7922-7927, 2005; Dongre, A. R. et al. Eur JImmunol 31, 1485-1494, 2001; Nedjic, J., et al., Curr Opin Immunol 21,92-97, 2009). There is also increasing evidence that the class I antigenprocessing machinery affects this endogenous pathway for class IIprocessing and presentation (Jaraquemada et al., J. Exp. Med.172:947-954, 1990; Loss et al., J. Exp. Med. 178:73-85, 1993; Oxenius,A. et al., Euro. J. Immunol. 25:3402-3411, 1995). Tewari et al. haveidentified two I-E^(d) restricted epitopes from the influenza HA and NAproteins that are processed by TAP and the proteasomal machinery andloaded onto recycling MHC class II molecules (Nature Immunol. 6:287-294,2005).

Plasmid expression vectors: Detection of overexpressed TAPs or MTI-PAAis determined by PCR, immunoprecipitation, Western blot,immunofluorescence and/or IHC. Immuno-detection makes use ofcommercially available antibodies (see Examples). Intracellularlocalization is evaluated by indirect immunofluorescence and subcellularfractionation using commercially available methodologies (e.g., NE-PER,Pierce, Inc.). Preparation of VACV vectors may be accomplished by theordinary skill artisan in accordance with standard methodologies.

Levels of antigen presentation are assessed by several methods: flowcytometry using purified p/MHC specific antibodies isolated fromhybridomas and conjugated to PE or APC (Molecular Probes AntibodyStaining Kits); co-incubation of transfected APCs with peptide-specificT cells and assessing immune recognition by IFNγ ELISPOT assay orintracellular cytokine staining.

One or more epitope tags may be used to aid in the detection of a fusionpolypeptide encoded by a nucleic acid molecule of this disclosure whencontained in a plasmid or viral vector, wherein the nucleic acidmolecule is operably linked to an expression control sequence.

Antigenic peptides (Class I or II restricted) arise when proteins orfragments thereof undergo proteasomal processing. The peptides generatedcan then bind to MEW (HLA) molecules and can elicit protective immunityif various additional parameters are met (Gilchuk et al., J. Clin.Investig. 123:1976-1987, 2013), such as (1) HLA binding affinity; (2)peptide/HLA stability; (3) interactions with molecular chaperones(TAP/HLA-DM) (Yin et al., J. Immunol. 189:3983-3994, 2012); (4) strengthof the TCR:peptide:MHC complex interaction; (5) antigen expression leveland kinetics; (6) cellular localization; and (7) T cellrecognition/peptide immunogenicity. In certain embodiments, peptideselicit multi-functional T cell responses and generate long-lived memorycells that protect the host from infectious disease or from developingcancer.

EXAMPLES Example 1 Nucleic Acid Molecules Comprising a MultiplexTranslation Initiation Sequence

FIG. 1 depicts an exemplary construction of a nucleic acid-basedexpression vector incorporating the MTI (based on FGF2 upstreammultiplex translation initiation from CUG start codons) technology asdisclosed herein. Expression vectors may comprise a plasmid vector ormay comprise a nucleic acid molecule encoding peptide antigenic epitopesincorporated into one or more viral delivery systems. A first series ofplasmid expression vectors encoding (a) MTI-PAA (PAA=Peptide AntigenArray; see FIG. 1C), (b) codon-optimized and epitope tagged TAP1, and(c) codon-optimized TAP2. These MTI-PAA nucleic acid molecule-basedsystems may further include combinations of TAP1 and/or TAP2. The vectordesign allows the PAA portion to be easily excised as a cassette andreplaced with the sequence corresponding to any series of T-cellepitopes, enabling incorporation of additional epitopes from otherpathogens, cancer genes or HLA supertypes into the expressionvector—enhancing its utility as a “plug-and-play” platform.

In the context of MHC Class I peptides, expression vectors direct thesynthesis of a chimeric protein containing an NH₂ portion of hFGF2 (MTI)followed by a specific peptide antigen array (PAA) representative of theaforementioned antigenic peptides. We assembled a selection of peptidesinto a linear peptide antigen Array (PAA) and converted the same into anucleic acid expression vector. However, we hypothesized that proteaseprocessing requires the presentation of a complete protease processingsite. Therefore, in order to encourage or otherwise recapitulateefficient proteasome processing of the peptide sequences, we extendedthe amino acid sequence at their NH₂ and COOH ends by three or four ormore native/naturally occurring amino acids.

The expression vectors incorporate features that will enhance the amountof selected PAA/peptides (T cell epitopes) synthesized. Particularfeatures include (1) codon optimized DNAs, and (2) the ability tosynthesize four moles of MTI-PAA for every one mole of mRNA transcribed.Codon optimization of the PAA portion of the expression vectors usedherein will increase the translational efficiency of transcribed mRNAs.In some cases, codon optimization also provides a means by which tomolecularly distinguish (e.g., by PCR) vector encoded genes (e.g., TAP1and TAP2) from corresponding endogenous genes in preclinical as well asclinical evaluation.

Regarding above mentioned ability to synthesize four moles of MTI-PAAfor every one mole of mRNA transcribed, expression system takesadvantage of the fact the human FGF2 gene is capable of directing thesynthesis of four polypeptides from one mRNA via the recognition ofthree unconventional CUG translation initiation codons and one classicalAUG codon. The three CUG codons initiate translation of three co-linearamino terminally extended versions of 18 kDa FGF2 that initiatestranslation at a downstream AUG codon. The portion of FGF2 included inthe vector described above retains the three CUG codons as well as theone AUG codon. Therefore, the vector provides four moles of a particularPAA for every one mole of mRNA.

An additional feature of the FGF2 portion of the expression vectors isthe ability to evaluate the efficiency of cytosolic versus nuclearproteasome processing. It is well established that proteasome processingprovides peptide substrates for MHC Class I mediated cell surfacepresentation. It is also appreciated that proteasomes differ incomposition, where, for example, immune proteasomes are distinct fromnon-immune (normal) proteasomes. Further, it is recognized, although notwell characterized, that nuclear proteasomes appear different fromcytosolic proteasomes.

FGF2 translation products initiated with CUG codons are targeted to thenucleus while the translation product initiated at the AUG codon iscytosolic (Data not shown). Accordingly, FGF2 sequences are modified(site specific or deletion mutagenesis) to exclusively express eithercytosolic or nuclear localized MTI-PAA chimera.

Example 2 Expression of Poly-Antigen Array Epitopes

TAP1, TAP2, and PolyStart™ MTI-PAA gene expression in transfected COS-1cells have been observed by PCR (data not shown). The pJ603 expressionplasmid was selected for these experiments. The CMV and/or SV40 latepromoter contained in the plasmid drives transcription of downstreamcoding sequence and (in some cases) sequence deemed important formaintaining mRNA structure and appropriate recognition of CUG/AUGtranslation initiation codons. Accordingly, we have designed/prepared afirst series of plasmid expression vectors encoding (a) MTI-PAA(smallpox peptides), (b) Tap1 only, (c) Tap2 only, (d) Tap1-IRES-Tap2,(e) Tap1 plus MTI-PAA, and (f) Tap2 plus MTI-PAA. A VSVG peptide-tagepitope tag was included at the COOH-terminal end of the MTI-PAA.

The expression vectors have been designed so that the PAA portion of theMTI-PAA can be easily excised and replaced with any codon-optimized DNAsequence encoding any PAA corresponding to any series of T-cell peptideepitopes (or full length/truncated protein), enabling us to rapidlyincorporate additional peptide epitopes such as VEEV E3 or E2, cancerepitopes such as breast cancer related epitopes (e.g., Her2/neu, folatereceptor alpha, NY-ESO-1, MIF, GnRh or GnRHR), or peptide epitopesspecific to any pathogen(s) or targeted cancer cell protein into ourexpression vector.

In addition, the nucleic acid sequences encoding TAP1, TAP2, and PAA,were prepared synthetically and in context with a preferred codonutilization algorithm. Codon optimization provides enhanced translationefficiency consistent with the preferred codon bias of Homo sapienswhile simultaneously minimizing potential cis-acting mRNA sequences thatcould also negatively impact translation efficiency. In this example,the FGF2 (MTI) portion was not codon optimized to preserve sequencedependent mRNA structure that may be needed to mediate translation fromunconventional (i.e., CUG) translation initiation codons in a mannermechanistically consistent with internal ribosome entry. This strategywill support higher levels of expression and thereby provide a higherlevel of corresponding antigenic polypeptide. This in turn is expectedto result in higher proteasome mediated peptide processing andconsequently higher TAP mediated peptide presentation to MHC andsubsequent cell surface presentation.

The unique aspects of the codon-refined synthetic DNAs encoding TAP1 andTAP2 have been utilized to design PCR primer pairs that distinguishcDNAs derived from endogenous mRNA transcripts from cDNA derived fromvector transcribed mRNA. The primer pairs were then used in PCRreactions using template DNA obtained from three sources: codon refinedplasmid DNA for TAP1 and TAP2; cDNA template from non-transfected COS-7and PANC-1 cells; and cDNA template from COS-7 cells transfected withTAP1 and TAP2 plasmid DNA.

COS-1 cells (70% confluent) were transfected in 60 mm plates using theFugene HD reagent (Promega, Inc.). Approximately 40 hourspost-transfection, total RNA was prepared. The total RNA was treatedwith DNase free RNase and cDNA was generated using M-MLV reversetranscriptase. HotStart Polymerase (Qiagen, Inc.) was used in the PCRreaction as follows: 95° hold, 95° 15′, [95° 30″, 54/57° 30″, 72°1′30″]_(40x) 72° 5′, 12° hold; +/−Q solution.

The results demonstrated that the PCR primers designed to detectendogenous TAP1 and TAP2 do not recognize plasmid DNAs containing codonrefined TAP1 or TAP2 (data not shown). In comparison, PCR primer pairsthat are specific to the synthetic codon refined DNAs do prime. Inaddition, TAP1 primers do not cross-prime with TAP2 templates.Similarly, using total RNA prepared from non-transfected COS-7 andPANC-1 cells, PCR primer pairs recognizing endogenous TAP1 and TAP2 donot recognize cDNA derived from vector encoded mRNA but do recognizecDNA templates derived from endogenous TAP1 and TAP2 mRNA (data notshown). Finally, cDNA prepared from mRNA derived from COS-7 cellstransfected with plasmid expression vectors containing codon preferredTAP1 and TAP2 sequences distinguished endogenous from transfected mRNAs(data not shown). These PCR conditions were used in order tofollow/confirm TAP1 and TAP2 expression following presentation oftherapeutic plasmid DNAs or following infection with recombinantvaccinia virus.

Further, since both TAP1 and TAP2 contain internalized non-cleavedsignal sequences, leaving an exposed cytosolic NH2 portion of TAP1 andan exposed ER-lumen localized NH2 portion of TAP2, our syntheticconstructs incorporate commonly used NH2-terminal epitope tags for thedifferential detection of vector encoded, as opposed to endogenous, TAP1and/or TAP2. The NH2-terminal TAP1 tag is V5 and the NH2-terminally tagfor TAP2 is AU5. Commercially available antibodies were used to detectexpression and intracellular localization by immunoprecipitation,immunoblotting, and immunofluorescence.

Protein expression of both TAP1 and MTI-PAA was confirmed byimmunoprecipitation (FIG. 2). Codon-optimized TAP2 protein expressionwas problematic and we are recreating the vectors using native humanTAP2 sequence. Immunofluorescence data demonstrate the cytosolicexpression of TAP1 using both anti-V5 and anti-TAP1 antibodies (FIG. 3).Control experiments characterizing full length expression of MTI-PAA wasalso confirmed by immunofluorescence staining of transfected COS cellsusing an anti-FGF2 Ab targeting the amino-terminus (data not shown) andan anti-VSV Ab directed against the C-terminal portion of MTI-PAA (FIG.4). These data also demonstrate that MTI-PAA is predominantly localizedto the nucleus. FIG. 2 also illustrates that MTI-PAA expression isenhanced in the presence of the proteosomal inhibitor MG132 (10 nM). IPdata from transfected HEK cells also indicates increased levels ofMTI-PAA in the presence of MG132 (FIG. 5) as well as detection of allfour predicted translation products of the MTI-PAA (FIG. 5).

Example 3 Expression and Immune Recognition of Poly-Antigen ArrayEpitopes

FIG. 6 demonstrates that peptide-specific T cells (from peptidevaccinated mice) recognize THP-1 cells transfected (Lipofectamine LTX,Invitrogen) with PolyStart™ MTI-PAA, confirming PAA-driven expression,processing, and presentation of immunogenic peptides. Furthermore, asillustrated in FIG. 7, co-expression of TAP1 in HEK PAA-transfectedcells influences antigen recognition by T cells.

Example 4 Murine Peptide Reactivity

Peptide-specific T cell responses from HLA-A2 transgenic miceintradermally vaccinated with Dryvax® were detected using IFNγ ELISPOT.Positive responses are defined as ≥2 fold increase in spot count overbackground with p<0.05 and peptides exhibiting consistent reactivity inmultiple mice are shown in Table 1. These same seven peptides wererecognized by T cells from two different strains of HLA-A2 mice(chimeric A2/K^(b) on a BL/6×Balb/c F1 background and chimeric A2/D^(d)on a BL/6×CBA F1 background), increasing confidence in the immunologicrelevance of these peptides following smallpox immunization

TABLE 1 Positive Peptides From HLA-A2 Mice Peptide Protein SEQ No.Sequence Source Description ID NO. 16 VLSLELPEV D13L Virion Coat 24(127) Protein 19 KIDYYIPYV E2L Hypothetical 25 (068) Protein 22SLSNLDFRL F11L Unknown 26 (058) Function 25 ILMDNKGLGV F1L Apoptosis 27(048) Inhibitor 28 ILDDNLYKV G5R Viral 28 (091) Morphogenesis 30KLLLGELFFL J3R Poly(A) 29 (104) Polymerase 33 GLLDRLYDL O1L Unknown 30(079) Function

Example 5 Peptide Immunogenicity Testing

HLA-A2 transgenic mice (n=3/group) were immunized twice with 10 μg ofthe individual peptides listed in Table 1. A2 peptides were emulsifiedin IFA along with 100 μg CpG1826 and 140 μg HBV core antigen (SEQ IDNO.: 31; TPPAYRPPNAPIL). Immune responses against peptide-pulsed targetcells were readily detectable four weeks after the second vaccination(FIG. 8). Mice immunized with a combination of peptides had detectableresponses 4-5 months (FIG. 9) after the second immunization. IFN-γELISPOT responses to individual peptides range from 2 to 19-foldincrease over background with each peptide exhibiting a consistentmagnitude of response (data not shown). These results demonstratesuccessfully elicited A2-restricted murine T cell responses to epitopesidentified from infected human cell lines, thus the model systemrecapitulates the antigen processing and presentation of a natural humaninfection.

Example 6 Verification of Murine Model Epitopes in Human Vaccines

Immune responses against a peptide pool (all 7 peptides from Table 1)were tested in 83 HLA-A2 supertype positive, human subjects who hadreceived the smallpox vaccine 1-4 years previously. 42.2% of thesubjects (35/83) had positive IFNγ ELISPOT responses to the peptide pool(Spot count ≥1.5 times that of background wells with t-test p<0.05).Responses ranged from 1.5 to 6.2-fold increase over background.Furthermore, 20.4% of an additional 54 HLA-A2 supertype positivesubjects had detectable immune responses against one or more of theindividual. Some peptides were not recognized by any of the subjects,while other peptides were recognized by multiple subjects (data notshown). These results support the use of the HLA transgenic mice toidentify relevant peptides recognized by human vaccine recipients.

Example 7 Efficacy of Identified Peptides

Mice (n=5) were vaccinated subcutaneously on the right flank with fourCTL peptides (#22, #25, #28, #30 from Table 1) 140 μg of the HBV Thelper epitope, and 100 μg of CpG 1826 emulsified in IFA. Unvaccinatedmice (n=5) served as controls. Three weeks after immunization, mice werechallenged intranasally with 1×10⁶ pfu Vaccinia Western Reserve.Survival, weight loss, and clinical symptoms of illness were monitoreddaily. Mice losing 25% of their body weight were euthanized.

Survival data are presented in FIG. 10. Peptide-vaccinated mice werecompletely protected from lethal challenge. In contrast, all of theunvaccinated mice developed severe clinical symptoms (ruffled fur,hunched posture, loss of mobility) and 80% of them succumbed to theinfectious challenge.

Example 8 Epitope Identification in Additional Pathogens

Methods of this disclosure can be used to create a nucleic acid moleculevaccine that encodes antigenic peptide epitopes from seven HLA class Isupertypes (A1, A2, A11, A24, B7, B27, B44) and three HLA-C alleles(Cw*0401, Cw*0602, Cw*0702), which would protect across a worldwidepopulations on average of >96% and is obtainable even withoutconsidering peptide binding promiscuity. Frequencies and resources fordetermine HLA class haplotypes are described in Robinson et al. NucleicAcids Res. 41:D1222, 2012.

Example 9 Her2/Neu+ Cancer Vaccine Compositions

The following example illustrates a peptide-based and a nucleicacid-based immunization composition for treating cancers thatoverexpress a HER2/neu marker protein such as HER2/neu overexpressingbreast cancer.

Compositions are prepared containing one or more HLA Class I and/orClass II antigenic T-cell peptide sequence generated by computeralgorithms or identified by testing human patient samples. For example,computer-based predictions identified a panel of 84 Class II HLA-DRbinding epitopes (Kalli et al., Cancer Res 68:4893, 2008; Karyampudi etal., Cancer Immunol Immunother 59:161, 2010). A pool containing fourHLA-DR epitopes are immunogenic, naturally processed, and cover about84% of Caucasians, African Americans and Asians (Hardy-Weinbergequilibrium analysis). The amino acid sequence of four individualpeptide epitopes includes NLELTYLPTNASLSF (SEQ ID NO.: 32),HNQVRQVPLQRLRIV (SEQ ID NO.: 33), LSVFQNLQVIRGRIL (SEQ ID NO.: 34), andPIKWMALESILRRRF (SEQ ID NO.: 35). Compositions containing these fourHLA-DR peptides are described in the Phase I Clinical Trial entitled “APhase I Trial of a Multi-Epitope HER2/neu Peptide Vaccine for PreviouslyTreated HER-2 Positive Breast Cancer”. In the trial, 500 μg of eachpeptide generate an immune response in up to about 90% of breast andovarian cancer patients (Knutson et al., J. Clin. Invest. 107:477, 2001;Disis et al., J. Clin. Oncol. 20:2624, 2002).

The peptide or nucleic acid vaccine compositions may also contain one ormore HLA Class I antigenic CD8⁺ T-cell antigenic epitope in combinationwith one or more HLA Class II epitopes. For example, the composition caninclude the epitopes as described in U.S. Provisional Application No.61/600,480 entitled “Methods and materials for generating CD8⁺ T-cellshaving the ability to recognize cancer cells expressing a Her2/neupolypeptide.” An exemplary amino acid sequence is SLAFLPESFD (SEQ IDNO.:36)(amino acids 373-382).

An HLA Class I antigenic epitope may be modified by extending at its NH2and/or COOH end by one or more naturally occurring amino acid. Such anextension may recapitulate an endogenously recognized naturallyprocessed polypeptide sequence involved in proteolytic processingthrough a cells proteasome mediated degradation pathway, which mayinclude sequences that promote ubiquitination. Accordingly, aplasmid-based nucleic acid immunization composition can incorporate thecorresponding DNA coding sequences, which may include nucleic acidsequences reflecting natural NH2 and/or COOH terminal extensions, andwhich may incorporate linker regions positioned between the codingsequences of each peptide.

An exemplary vaccination strategy is to administer as a prime-and-boost,i.e., sequentially delivering one or more in a series of peptide vaccinecomposition(s), followed by one or more in a series of nucleic-acid(e.g., plasmid or viral) vaccine compositions, or vice versa. In oneaspect, a peptide immunization composition is administered prior toadministration of a nucleic acid immunization composition. In anotheraspect, the nucleic acid immunization composition is administered priorto administration of the peptide immunization composition.

In addition, some patients may have a better outcome if prior toadministering a vaccine composition, the patient receivescyclophosphamide, or a similar composition, orally for 1 week followedby a 1 week rest, and then another week of cyclophosphamide treatment.About 7-10 days following cyclophosphamide treatment, patients arevaccinated intradermally, intramuscularly, or ex vivo using isolatedcells. The peptides may be HLA-DR related and the nucleic acid mayencode one or more HLA-Class I antigenic cytotoxic T-cell epitopes.

Compositions are prepared containing one or more HLA Class I and/orClass II antigenic T-cell peptide sequences from computer algorithms ortesting human patient samples. The vaccine compositions may contain anadjuvant, such as GM-CSF (e.g., 125m/injection).

Example 10 Folate Receptor Alpha and Cancer Vaccine Compositions

This Example illustrates a combined or sequentially administered nucleicacid-based composition and peptide-based composition for treating afolate receptor alpha expressing cancer. Folate receptor alpha isoverexpressed in a number of cancers such as ovarian, primaryperitoneal, lung, uterine, testicular, colon, renal, HER2/neu⁺ breast,and triple negative breast cancer (Zhang et al., Arch. Pathol. Lab. Med.p 1-6, 2013; Weitman et al., Cancer Res. 52:3396, 1992; O'Shannesst etal., SpringerPlus 1:1, 2012; Kelemen, Int. J. Cancer 119:243, 2006; U.S.Pat. No. 8,486,412).

In one aspect, the vaccine composition contains one or more antigenicfolate receptor alpha HLA Class II and/or HLA Class I antigenic peptideepitopes as described in a Phase I Clinical Trial entitled “A Phase ITrial of the Safety and Immunogenicity of a Multi-epitope FolateReceptor Alpha Peptide Vaccine in Combination with Cyclophosphamide inSubjects Previously Treated for Breast or Ovarian Cancer.” A vaccinecomposition comprising one or more HLA Class II folate receptor alphaantigenic T-cell peptides comprising for example peptides FR30 (aminoacid sequence RTELLNVCMNAKHHKEK (SEQ ID NO.: 37)), peptide FR56 (aminoacid sequence QCRPWRKNACCSTNT (SEQ ID NO.: 38)), peptide FR76 (aminoacid sequence KDVSYLYRFNWNHCGEMA (SEQ ID NO.: 39)); peptide FR113 (aminoacid sequence LGPWIQQVDQSWRKERV (SEQ ID NO.: 40)), and peptide FR238(amino acid sequence PWAAWPPLLSLALMLLWL (SEQ ID NO.: 41)), and mayoptionally be formulated with one or more similarly identified HLA ClassI folate receptor alpha antigenic T-cell peptides. It is of note thatone or more Class I antigenic T-cell epitopes may be contained withinthe sequence of a HLA Class II epitope. For example, the Class IIpeptide FR56 and the peptide sequence of FR238 containing the HLA ClassI epitope of folate receptor alpha peptides 245-253. Composition mayalso be formulated with an adjuvant such as GM-CSF and/or combined withprior treatment with an immune-regulatory substance such ascyclophosphamide or denileukin diftitox (Ontak).

The plasmid-based nucleic acid immunization composition incorporates thecorresponding DNA coding sequences for a folate receptor alpha HLA ClassII and/or HLA Class I antigenic T-cell peptide. The DNA may includenucleic acid sequences reflecting natural NH2 and/or COOH terminalextensions which are thought to recapitulate the naturally occurringprotease processing site for proteasome mediated degradation. Further,the DNA may incorporate linker regions positioned between the codingsequences of each peptide.

A matched vaccination strategy is one in which the peptides and nucleicacid encoded sequences are derived from the same cancer related TAA. Thematched vaccination strategy includes administering respectivecompositions as a prime-and-boost, i.e., sequentially delivering one ormore of a series of folate receptor alpha antigenic T-cell peptidevaccine composition(s), followed by one or more in a series of folatereceptor alpha peptide derived nucleic-acid vaccine compositions (e.g.,plasmid or viral). In one aspect, the peptide immunization compositionis administered prior to administration of a nucleic acid immunizationcomposition. In another aspect, the nucleic acid immunizationcomposition is administered prior to administration of the peptideimmunization composition. The peptides may be HLA-DR related and thenucleic acid may encode one or more HLA-Class I antigenic cytotoxicT-cell epitopes.

Additional cancer TAAs may be included in the vaccine compositions,including antigenic T cell peptides from insulin-like growth factorbinding protein 2 (Kalli et al., Cancer Res. 68:4893, 2008),carcinoembryonic antigen (Karyampudi et al., Cancer Immunol. Immunother.59:161, 2010), and PD-1 antagonists (Krempski et al., J. Immunol.186:6905, 2011).

Compositions are prepared containing one or more HLA Class I and/orClass II antigenic T-cell peptide sequences from computer algorithms ortesting human patient samples. The vaccine compositions may contain anadjuvant such as GM-CSF (e.g., 125m/injection). In addition, somepatients may have a better outcome if prior to administering a vaccinecomposition, the patient receives cyclophosphamide, or a similarcomposition, orally for 1 week followed by a 1 week rest, and thenanother week of cyclophosphamide treatment. About 7-10 days followingcyclophosphamide treatment, patients are vaccinated intradermally,intramuscularly, or ex vivo using isolated cells.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, including butnot limited to U.S. Patent Application No. 61/954,588, filed Mar. 17,2014, are incorporated herein by reference, in their entirety. Aspectsof the embodiments can be modified, if necessary to employ concepts ofthe various patents, applications and publications to provide yetfurther embodiments.

In general, in the following claims, the terms used should not beconstrued to limit the claims to the specific embodiments disclosed inthe specification and the claims, but should be construed to include allpossible embodiments along with the full scope of equivalents to whichsuch claims are entitled. Accordingly, the claims are not limited by thedisclosure.

1. A method of eliciting an immune response, comprising administering toa human subject an effective amount of a population of immune cellscomprising immune cells containing a chimeric nucleic acid molecule,wherein the chimeric nucleic acid molecule comprises a multiplextranslation initiation (MTI) sequence comprising from two to about fivetranslation initiation sites operatively linked in frame to a nucleicacid molecule encoding a fusion protein comprised of a plurality ofantigenic peptides, wherein at least one of the MTI translationinitiation sites is a non-AUG translation initiation site and the MTIallows the production of more than one mole of fusion protein per moleof mRNA, thereby eliciting an immune response.
 2. The method of claim 1,wherein the MTI comprises one, two, three, or four non-AUG translationinitiation sites.
 3. The method of claim 2, wherein one, two, three, orfour of the non-AUG translation initiation sites are CUG translationinitiation sites.
 4. The method of claim 3, wherein the MTI comprises anAUG translation initiation site downstream of the one, two, three, orfour CUG translation initiation sites.
 5. The method of claim 4, whereinthe MTI comprises one or two nuclear localization domains locatedupstream of the AUG translation initiation site and downstream of atleast one CUG translation initiation site.
 6. The method of claim 1,wherein the MTI comprises a 5′-portion of a human FGF2 gene, wherein the5′-portion of the human FGF2 gene contains an FGF2 AUG translationinitiation site and about 123 nucleotides to about 385 nucleotidesupstream of the FGF2 AUG translation initiation site that is in framewith the nucleic acid molecule encoding the fusion protein.
 7. Themethod of claim 6, wherein the MTI further comprises from about 15nucleotides to about 45 nucleotides downstream of the FGF2 AUGtranslation initiation site.
 8. The method of claim 6, wherein the5′-portion of the human FGF2 gene encodes a polypeptide having at least90% sequence identity to any one of the amino acid sequences set forthin SEQ ID NOS.:8-12, or encodes a polypeptide as set forth in any one ofSEQ ID NOS.:8-12.
 9. The method of claim 1, wherein the MTI sequence hasat least 90% sequence identity to a nucleotide sequence as set forth inany one of SEQ ID NOS.:1-6, 95, or
 96. 10. The method of claim 1,wherein the encoded fusion protein comprises from two to about tenantigenic peptides.
 11. The method of claim 10, wherein two to about tenof the antigenic peptides are each different from each other.
 12. Themethod of claim 1, wherein the antigenic peptide comprises atumor-associated antigen or a pathogen-related antigen.
 13. The methodof claim 12, wherein the tumor-associated antigen comprises an antigenassociated with breast cancer, triple negative breast cancer,inflammatory breast cancer, ovarian cancer, uterine cancer, colorectalcancer, colon cancer, primary peritoneal cancer, testicular cancer,renal cancer, melanoma, glioblastoma, lung cancer, prostate cancer, orany combination thereof.
 14. The method of claim 12, wherein thepathogen-related antigen comprises an antigen from a virus, parasite, orbacteria.
 15. The method of claim 10, wherein one or more of theantigenic peptides are an HLA Class I antigenic peptide, an HLA Class IIantigenic peptide, an HLA Class II antigenic peptide comprising anembedded HLA Class I antigenic peptide, or any combination thereof. 16.The method of claim 1, wherein the encoded fusion protein comprises anamino acid cleavage sequence amino-terminal to one or more of theencoded polypeptide components of the fusion protein, wherein the aminoacid cleavage sequence comprises a 2A peptide from porcine teschovirus-1(P2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), orfoot-and-mouth disease virus (F2A).
 17. The method of claim 1, whereinthe encoded fusion protein comprises a secretion signal amino acidsequence, a membrane localization amino acid sequence, an endosometargeting sequence, a dendritic cell targeting amino acid sequence, orany combination thereof.
 18. The method of claim 1, wherein the encodedfusion protein comprises: (a) a VSVG signal amino acid sequence asencoded by the polynucleotide of SEQ ID NO.:43 that is operably linkedin frame to and disposed between the MTI sequence and the nucleic acidmolecule encoding the fusion protein, optionally comprising a nucleicacid molecule encoding an amino acid cleavage sequence operably linkedin frame to and disposed between the MTI sequence and the nucleic acidmolecule encoding the VSVG signal amino acid sequence; (b) a VSVGtrafficking amino acid sequence as encoded by the polynucleotide of SEQID NO.:47 or 48 that is operably linked in frame to the MTI sequence;(c) a VSVG membrane localization amino acid sequence as encoded bynucleotides 457 to 516 of SEQ ID NO.:57 that is operably linked in frameto the MTI sequence; (d) a VSVG trafficking amino acid sequence andmembrane localization amino acid sequence as encoded by thepolynucleotide of SEQ ID NO.:57 that is operably linked in frame to theMTI sequence; (e) any one of (a)-(d) further comprising a dendritic celltargeting amino acid sequence as encoded by the polynucleotide of SEQ IDNO.:45; or any one of (a) or (c)-(e) further comprising an intracellulartrafficking sequence.
 19. The method of claim 1, wherein the chimericnucleic acid molecule is: (a) an mRNA molecule; (b) a DNA molecule; (c)a DNA or an RNA molecule contained in a vector and operably linked to anexpression control sequence.
 20. The method of claim 19, wherein thevector of subpart (c) of claim 21 comprises: (a) a plasmid vectorcomprised of DNA, wherein the chimeric nucleic acid molecule is a DNAmolecule contained in the DNA plasmid vector, (b) a viral vectorcomprised of DNA, wherein the chimeric nucleic acid molecule is a DNAmolecule contained in the DNA viral vector, or (c) a viral vectorcomprised of RNA, wherein the chimeric nucleic acid molecule is an RNAmolecule contained in the RNA viral vector.
 21. The method of claim 20,wherein the vector comprises a viral vector selected from a rhabdoviral,adenoviral, herpesviral, poxviral, or retroviral vector.
 22. The methodof claim 1, wherein the chimeric nucleic acid molecule is formulated asa composition comprising a therapeutically acceptable carrier orexcipient.
 23. The method of claim 1, wherein the elicited immuneresponse comprises a cellular immune response.
 24. The method of claim1, further comprising administering an effective amount of an antigenicpeptide immunization composition.
 25. The method of claim 24, whereinthe chimeric nucleic acid molecule encodes one or more of the sameantigenic peptides of the antigenic peptide composition.
 26. The methodof claim 25, wherein: (a) the population of immune cells comprisingimmune cells containing the chimeric nucleic acid molecule and theantigenic peptide immunization compositions are administeredsimultaneously; (b) the population of immune cells comprising immunecells containing the chimeric nucleic acid molecule and the antigenicpeptide immunization compositions are administered sequentially; (c) thepopulation of immune cells comprising immune cells containing thechimeric nucleic acid molecule is administered from about 1 hour toabout 8 weeks after administering the antigenic peptide immunizationcomposition; (d) the antigenic peptide immunization composition isadministered from about 1 hour to about 8 weeks after administering thepopulation of immune cells comprising immune cells containing thechimeric nucleic acid molecule; (e) any one of (a)-(d), wherein theimmune cells are autologous to the human subject or allogeneic to thehuman subject; or (f) any one of (a)-(e), wherein the immune cells are Tcells.
 27. The method of claim 26, further comprising one or moreadditional administrations of an effective amount of the antigenicpeptide immunization composition and/or the chimeric nucleic acidmolecule after the first administration of the antigenic peptideimmunization composition and/or the chimeric nucleic acid molecule. 28.The method of claim 1, wherein the population of immune cells comprisingthe immune cells containing the chimeric nucleic acid molecule isadministered intradermally.
 29. The method of claim 12, wherein beforeor after administering the population of immune cells comprising thechimeric nucleic acid molecule encoding a fusion protein comprised of aplurality of tumor-associated antigens, the human subject is treatedwith surgery, chemotherapy, radiation therapy, antibody therapy,immunosuppressive therapy, or any combination thereof.
 30. The method ofclaim 12, wherein before or after administering the population of immunecells comprising the chimeric nucleic acid molecule encoding a fusionprotein comprised of a plurality of tumor-associated antigens, the humansubject is administered cyclophosphamide, trastuzumab, anti-PD1,anti-PDL1, anti-CTLA4, or any combination thereof.